Nitrobacter capable of resisting nitrous acid and ammonia nitrogen and application of nitrobacter

Abstract

The invention discloses nitrous acid and ammonia nitrogen resistant nitrobacter (Nitrobacter sp.) TKz-YC01, the preservation number of the nitrous acid and ammonia nitrogen resistant nitrobacter (Nitrobacter sp.) TKz-YC01 is GDMCC No: 65729, the nitrous acid and ammonia nitrogen resistant nitrobacter (Nitrobacter sp.) TKz-YC01 has been preserved in Guangdong Microbial Culture Collection Center on March 7, 2025, is called GDMCC for short, has the preservation address of Nitrobacter sp., and is classified and named as Nitrobacter sp., and the preservation name of the nitrous acid and ammonia nitrogen resistant nitrobacter (Nitrobacter sp. TKz-YC01 is Nitrobacter sp. The nitrobacter can effectively degrade nitrite, cyanate and / or ammonium salt in water, and the nitrobacter can still effectively remove nitrogen under the impact of 30 mM ammonium salt (ammonium chloride) and 50 mM nitrite (sodium nitrite).

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to a nitrobacter capable of resisting nitrous acid and ammonia nitrogen and application thereof. BACKGROUND

[0002] Nitrification is a key component of the nitrogen cycle process, and its core process is to oxidize ammonium to nitrate. This process plays a crucial role in the nitrogen cycle and denitrification process. Specifically, nitrification first oxidizes -3 valence ammonium to +3 valence nitrite, and then further oxidizes nitrite to +5 valence nitrate.

[0003] Nitrate is not only an important nitrogen source for many organisms, but also an indispensable electron acceptor in anaerobic respiration; in addition, the nitrite oxidation pathway is the only known biochemical pathway that can generate nitrate with +5 valence nitrogen.

[0004] Nitrite oxidation can be driven by classic nitrite oxidizing bacteria (NOB) or complete ammonia oxidation bacteria (comammox), but studies have shown that comammox is not a "full-time" nitrite oxidizing bacteria, and its activity depends on the presence of ammonium, and it is difficult to independently complete the conversion of nitrite to nitrate; Unlike comammox, which can oxidize nitrite by "borrowing ammonia nitrogen", NOB can directly use nitrite as the only nitrogen source and energy source for obligate chemolithoautotrophic growth, without the need for ammonium salt participation, and can proliferate without any external organic matter. As a key "gear" in the nitrification process in nature, NOB is widely distributed in various environments and plays an irreplaceable role in global nitrogen cycle.

[0005] And nitrous acid, as the main substrate of nitrifying bacteria, and high concentration of nitrous acid shows a certain inhibition to all nitrifying bacteria; in the water environment with high concentration of nitrite, both the aquatic animals (fish, etc.) living in it and the activity of nitrifying bacteria in the water environment will be inhibited, and the nitrifying bacteria cannot effectively degrade the nitrite in the water environment under this condition.

[0006] Therefore, there is an urgent need for a nitrifying bacteria capable of resisting high-concentration nitrous acid shock and ammonia nitrogen shock, so as to achieve better denitrification and environmental governance, and realize more stable nitrogen cycle. SUMMARY

[0007] The purpose of the present application is to overcome the above-mentioned shortcomings of the prior art, and to provide a nitrobacter capable of resisting nitrous acid and ammonia nitrogen and application thereof.

[0008] The first object of the present application is to provide a nitrobacter sp. Nitrobacter sp . ) TKz-YC01.

[0009] The second object of the present application is to provide an application of the above-mentioned nitrobacter sp.

[0010] The third object of the present application is to provide an application of the above-mentioned nitrobacter sp.

[0011] The fourth object of the present application is to provide an application of the above-mentioned nitrobacter sp.

[0012] The fifth object of the present application is to provide an application of the above-mentioned nitrobacter sp.

[0013] The sixth object of the present application is to provide an application of the above-mentioned nitrobacter sp.

[0014] The seventh object of the present application is to provide an application of the above-mentioned nitrobacter sp.

[0015] The eighth object of the present application is to provide a microbial inoculant.

[0016] In order to achieve the above-mentioned objects, the present application is realized by the following scheme: The present application provides a newly isolated nitrobacter sp.

[0017] Therefore, the present application claims the following content: A nitrobacter sp. Nitrobacter sp . ) TKz-YC01, the nitrobacter sp. Nitrobacter sp .The preservation number of TKz-YC01 is GDMCC No: 65729, which was preserved in Guangdong Microbial Culture Collection Center (GDMCC) on March 7, 2025, and the address of the preservation is: 5th floor, No. 59 Building, Guangzhou Martyrs' Park, 100 Xianlie Road, Guangzhou, and the classification name is Nitrobacter sp . , and the preservation name is Nitrobacter sp . TKz-YC01.

[0018] The application also claims the application of the above-mentioned Nitrobacter sp. Nitrobacter sp . TKz-YC01 in water body treatment.

[0019] Preferably, the water body treatment is to reduce the content of nitrite in the water body, reduce the content of cyanate in the water body, and / or reduce the content of ammonia nitrogen in the water body.

[0020] The application also claims the application of the above-mentioned Nitrobacter sp. Nitrobacter sp . TKz-YC01 in the preparation of a product for degrading nitrite.

[0021] The application also claims the application of the above-mentioned Nitrobacter sp. Nitrobacter sp . TKz-YC01 in the preparation of a product for degrading cyanate.

[0022] The application also claims the application of the above-mentioned Nitrobacter sp. Nitrobacter sp . TKz-YC01 in the preparation of a product for degrading ammonia nitrogen.

[0023] The Nitrobacter sp. TKz-YC01 claimed in the application can convert nitrite in the water body into nitrate, whether in a high-concentration nitrite environment or a low-concentration nitrite environment, and can still be effectively converted under the impact of 50 mM nitrite.

[0024] Therefore, the application also claims the application of the above-mentioned Nitrobacter sp. Nitrobacter sp . TKz-YC01 in the preparation of a product resistant to nitrite.

[0025] The application also claims the application of the above-mentioned Nitrobacter sp. Nitrobacter sp . TKz-YC01 in the preparation of a product resistant to ammonium salt.

[0026] Preferably, the Nitrobacter sp. Nitrobacter sp .) TKz-YC01 includes nitrobacterium (Nitrobacter winogradskyi) Nitrobacter sp . ) At least one of the living bacteria, the bacterial culture solution or the culture supernatant of TKz-YC01.

[0027] The present application also claims a microbial agent containing the above-mentioned nitrobacterium (Nitrobacter winogradskyi) Nitrobacter sp . ) TKz-YC01.

[0028] Compared with the prior art, the present application has the following beneficial effects: The present application provides a nitrobacterium (Nitrobacter winogradskyi) Nitrobacter sp . ) TKz-YC01 which is resistant to nitrite and ammonia nitrogen, the preservation number of the nitrobacterium (Nitrobacter winogradskyi) Nitrobacter sp . ) TKz-YC01 is GDMCC No: 65729, which has been preserved in the Guangdong Microbial Culture Collection Center (GDMCC) on March 7, 2025, the address of the preservation is: No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100, the classification name is Nitrobacter sp . , and the preservation name is Nitrobacter sp . TKz-YC01. The nitrobacterium can effectively degrade nitrite, cyanate and / or ammonium salt in water, and the nitrobacterium can still effectively denitrify under the impact of 30 mM ammonium salt (ammonium chloride) and 50 mM nitrite (sodium nitrite). BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 It is the phylogenetic tree graph in Example 1; Figure 2 It is the morphological identification result graph of the nitrobacterium Nitrobacter sp . TKz-YC01 in Example 1; A is the electron microscope observation result graph of 15000x; B is the electron microscope observation result graph of 5000x; Figure 3 It is the average nucleotide identity result graph of the nitrobacterium Nitrobacter sp . TKz-YC01 in Example 2; Figure 4 It is the average amino acid identity result graph of the nitrobacterium Nitrobacter sp . TKz-YC01 in Example 2; Figure 5 It is the result graph of Example 3 Nitrobacter sp .Figure of test results of TKz-YC01 nitrite consumption; A is the nitrite level of each rotation speed of the conical flask culture process every day; B is the nitrate level of each rotation speed of the conical flask culture process every day; C is the dissolved oxygen content in the conical flask of each rotation speed; Figure 6 Figure of test results of ammonia nitrogen degradation in Example 3; A is the nitrite concentration result chart of the ammonia nitrogen degradation test experiment; B is the nitrate concentration result chart of the ammonia nitrogen degradation test experiment; C is the ammonium concentration result chart of the ammonia nitrogen degradation test experiment; Figure 7 Figure of cyanate degradation test results in Example 3; A is the experimental results of the control group; B is the experimental results of the experimental group; Figure 8 Figure of ammonium salt tolerance test results in Example 3; A is the nitrite concentration result chart; B is the nitrate concentration result chart; Figure 9 Figure of nitrite tolerance test results in Example 3. DETAILED DESCRIPTION

[0030] The present application will be further described below in conjunction with the accompanying drawings and specific examples, which are only used to explain the present application and are not intended to limit the scope of the present application. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

[0031] Example 1 Screening and identification of nitrobacterium TKz-YC01 I. Isolation of strains Groundwater samples were collected from Longmen Village, Guilin City, Guangxi Zhuang Autonomous Region (25.2118°N, 110.3676°E). 1 mL of the groundwater sample was inoculated into a capped Erlenmeyer flask containing 100 mL of 1×AOM sterile enrichment medium for culture. The components of the 1×AOM sterile enrichment medium included (1) a culture stock solution: 0.05 g / L KH2PO4, 0.075 g / L KCl, 0.05 g / L MgSO4·7H2O, 0.584 g / L NaCl, and 0.147 g / L CaCl2·2H2O. 2O; (2) 2nM NaHCO3; (3) Trace element solutions: 34.4mg / L MnSO4·1H2O, 50.0mg / L H3BO3, 70.0mg / L ZnCl2, 72.6mg / L Na2MoO4·2H2O, 20.0mg / L CuCl2·2H2O, 24.0mg / L NiCl2·6H2O, 80.0mg / L CoCl2·6H2O and 1.00g / L FeSO4·7H2O; (4) 5mM NaNO2 substrate.

[0032] Next, the capped conical flasks were placed in the dark at 28°C and 180 rpm. During the incubation process, the consumption of nitrite (NaNO2) was monitored. After the nitrite was consumed, 5 mM NaNO2 was added. Then, the flasks were transferred to fresh 1×AOM sterile enrichment medium at 1% (v / v) every month for 6 months.

[0033] After 6 months of culture, 0.1 mL of the culture medium was serially diluted (10-fold is one dilution, and a total of 5 dilutions were performed, i.e., 10^10 dilutions). -1 ~10 -5 The culture solutions were then serially diluted. 0.05 mL of each diluted culture solution was spread onto 1×AOM solid medium (1×AOM sterile enrichment medium with 1% agar) containing 1.0% (w / v) agar (Sangon Biotech, A100637-0500) and incubated at 28°C in the dark for 21 days. After incubation, a single colony was picked and dissolved in 0.01 mL of 1×AOM sterile enrichment medium and incubated at 28°C for 3 weeks. The consumption of NaNO2 substrate was examined. Strains in the NaNO2-consuming medium were used as positive samples and purified multiple times to obtain a pure culture of Nitrobacter. The pure culture of Nitrobacter contained a pure culture of Nitrifying Bacillus. Nitrobacter sp. TKz-YC01.

[0034] II. Purity Detection of Strains 1. Experimental Methods (1) 16S rRNA sequencing and phylogenetic analysis The DNA of the strain in the Nitrobacter pure culture liquid obtained in step one was extracted using the Tian Gen Bacterial Genomic DNA Extraction Kit (DP302) (nitrobacter Nitrobacter sp. The DNA of TKz-YC01 was amplified by PCR using the universal primers 8F (SEQ ID NO: 1) and U1492R (SEQ ID NO: 2).

[0035] The PCR product was purified using the TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0 kit (TaKaRa, item number 9761), and the purified PCR product was cloned using the TaKaRa Mighty TA-cloning Kit (TaKaRa, item number 6028). The cloning product was introduced into DH5a competent cells for transformation, and the transformed positive cells were sent to Guangzhou Jinweizhi Company for sequencing using M13F(-47) (SEQ ID NO: 3) and M13R(-48) (SEQ ID NO: 4). After removing the vector sequence, the 16S rRNA sequence of the strain was obtained.

[0036] The 16S rRNA sequence of the strain was analyzed by phylogenetic tree analysis using MEGA software with the 16S rRNA gene sequences of the Nitrobacter genus in the NCBI database.

[0037] (2) Strain purity detection The PCR product in step (1) was subjected to Sanger sequencing, and the Sanger sequencing results were recorded.

[0038] The bacterial liquid in the Nitrobacter pure culture liquid was inoculated into LB medium, BHI medium and TSB medium at a ratio of 1 / 1000 (v / v) respectively, and cultured at 28°C for 7 days. The turbidity of the culture medium was observed.

[0039] 2. Experimental results (1) 16S rRNA sequencing and phylogenetic tree analysis results The 16S rRNA sequence of the strain is shown in SEQ ID NO: 5.

[0040] 16S rRNA sequence (SEQ ID NO: 5):

[0041] Evolutionary tree diagram as follows Figure 1 As shown, the results indicate that the isolated strain (the bacteria in the pure culture of Nitrobacter) belongs to the genus Nitrobacter, and it is named Nitrifying Bacillus. Nitrobacter sp. TKz-YC01.

[0042] (2) Strains purity Sanger sequencing of the PCR products showed no overlapping peaks, indicating that there was no contamination from other microorganisms in the pure culture of Nitrobacter. Furthermore, no turbidity was observed when the pure culture of Nitrobacter was inoculated into three organic culture media, indicating that there was no contamination from other microorganisms in the pure culture of Nitrobacter.

[0043] III. Morphological identification of the strain 1. Experimental Methods The pure culture of Nitrobacter obtained in step one was inoculated onto 1×AOM solid medium and incubated at 28°C. Approximately 10 μL of the culture was then collected. 7 One bacterial sample (the precipitate size after centrifugation was between that of a rice grain and a soybean) was fixed into a 2 mL centrifuge tube containing 1.5 mL of glutaraldehyde (2.5%, wt%) and placed in a 4°C refrigerator for 24 h to obtain a fixed sample.

[0044] Next, the fixed sample (bacteria) was removed from the fixative and rinsed four times with 0.1M PBS for 10 minutes each time. After rinsing, the sample was fixed for 1 hour in the dark using a mixture containing 1% (wt%) osmium tetroxide and 2% (wt%) potassium ferricyanide (K3[Fe(CN)6]). Then, the liquid was removed and the sample was rinsed four times with deionized water for 10 minutes each time to obtain the post-fixed sample.

[0045] Next, the post-fixed sample was placed in 30% ethanol-50% ethanol-70% ethanol-80% ethanol-95% ethanol-anhydrous ethanol-anhydrous ethanol for dehydration in sequence for 10 minutes each time. Finally, it was dehydrated twice with 100% acetone for 10 minutes each time to obtain the dehydrated sample.

[0046] Then the dehydrated sample was transferred into a solution of acetone and embedding agent (SPI, 90529-77-4) mixed at a mass ratio of 3:1 for 1 h at 37°C, then into a solution of acetone and embedding agent (SPI, 90529-77-4) mixed at a mass ratio of 1:1 for 2 h at 37°C, then into a solution of acetone and embedding agent (SPI, 90529-77-4) mixed at a mass ratio of 1:3 for 2 h at 37°C, and then into pure embedding agent (SPI, 90529-77-4) for 8 h at 37°C to obtain the permeated sample.

[0047] Then the pure embedding agent (SPI, 90529-77-4) was poured into the embedding plate, the permeated sample was placed in the embedding plate, and the embedding agent was filled to the top, and then the embedding plate was placed in a 65°C oven for polymerization for 24 h to obtain the sample to be observed (resin block).

[0048] The resin block was cut into ultrathin sections of 70-90 nm using an ultramicrotome (Leica, Leica UC7), and then the ultrathin sections were stained with 2% ethanol uranium in the dark for 4 min, then rinsed with deionized water for 6 times (2 h each time), then stained with lead citrate in the dark for 4 min, rinsed with deionized water for 6 times (2 h each time), dried, and then observed using a JEM-1400F transmission electron microscope, and the observation results at magnifications of 5000x and 15000x were recorded.

[0049] 2. Observation results Nitrobacter Nitrobacter sp. The morphological identification results of TKz-YC01 are shown in FIGS. Figure 2 A in FIG. Figure 2 B in FIG. Figure 2 is the electron microscope observation result at 5000x.

[0050] The results show that the Nitrobacter Nitrobacter sp. TKz-YC01 cells contain a variety of typical structures, mainly including carboxysomes (CX) involved in carbon fixation, polyphosphates (PP) as phosphorus and energy storage substances, and plasma membrane intrusion systems (PMI) related to autotrophic growth; and the strain exhibits polymorphism characteristics, and various cell morphologies such as pear-shaped, avocado-shaped and elliptical-shaped can be seen.

[0051] Four, fatty acid identification of the strain 1. Experimental method The Nitrobacter pure culture obtained in step one was inoculated on 1x AOM solid medium by plate streaking method, and incubated at 28℃ for 1 month (30 days). The culture medium with colonies was sent to Guangdong Microbial Culture Collection Center, which was entrusted to use SHERLOCK 2.4 system for fatty acid identification.

[0052] 2. Experimental results The fatty acid identification results are shown in Table 1.

[0053] Table 1 Fatty acid identification results

[0054] Note: The peak at RT 3.3920 is a mixed peak, but most of it is 18:1 w8c.

[0055] The results show that the main fatty acid of the strain (Nitrobacter Nitrobacter sp. TKz-YC01) in the Nitrobacter pure culture is 18:1 w8c (isooleic acid), and 19:0 cyclo w8c and 16:0 (palmitic acid) also account for a high proportion. The main fatty acid composition of this strain is consistent with the analysis results of Nitrobacter strains in previous studies (Lipski A, Spieck E, Makolla A, Altendorf K. Fatty acid profiles of nitrite-oxidizing bacteria reflect their phylogenetic heterogeneity. Syst Appl Microbiol. 2001 Nov;24(3):377-84.), and no hydroxy fatty acid was detected. However, the isooleic acid content (52.8%) of this strain is significantly lower than that of other Nitrobacter strains (all higher than 72%).

[0056] V. Strain preservation The above-obtained Nitrobacter Nitrobacter sp. TKz-YC01 was preserved in Guangdong Microbial Culture Collection Center on March 7, 2025, referred to as GDMCC, with the preservation number GDMCC No: 65729, the preservation address being No. 59 Building, 5th Floor, Guangzhou Xianlie Middle Road 100 Courtyard, and the classification name being Nitrobacter sp. , and the preservation name being Nitrobacter sp. TKz-YC01 .

[0057] Example 2 Whole genome sequencing and new species identification of Nitrobacter TKz-YC01 I. Experimental method The genomic DNA of the nitrifying bacteria obtained in Example 1 was extracted using a TianGen Bacterial Genomic DNA Extraction Kit. Nitrobacter sp. The bacterial DNA of TKz-YC01 was detected for quality by a Nanophotometer and a Qubit, and the qualified sample was sent to Shanghai Meiji Bio for sequencing and genome assembly, obtaining the nitrifying bacteria Nitrobacter sp. The complete genomic information of TKz-YC01.

[0058] Based on the obtained complete genomic information, the average nucleotide identity (ANI) between TKz-YC01 and the reference strains was calculated by JSpeciesWS (https: / / jspecies.ribohost.com / jspeciesws / ). Nitrobacter sp. The average amino acid identity (AAI) between TKz-YC01 and the reference strains was calculated by the AAI Matrix Calculator of Kostas Lab (http: / / enve-omics.ce.gatech.edu / g-matrix / ). Nitrobacter sp. The DNA-DNA hybridization similarity (DDH) between TKz-YC01 and the reference strains was calculated by Genome-to-Genome Distance Calculator 3.0 (https: / / ggdc.dsmz.de / ggdc.php#), and ANI≥95%, AAI≥95% and DDH≥70% were taken as the threshold values for identifying new species. Nitrobacter sp. The DNA-DNA hybridization similarity (DDH) between TKz-YC01 and the reference strains was calculated by Genome-to-Genome Distance Calculator 3.0 (https: / / ggdc.dsmz.de / ggdc.php#), and ANI≥95%, AAI≥95% and DDH≥70% were taken as the threshold values for identifying new species. The reference strains include: Nitrobacter hamburgensis X14 (ASM1388v1), Nitrobacter sp. Nb-311A (ASM15290v1), Nitrobacter sp. NHB1 (ASM3696466v1), Nitrobacter vulgaris Ab1 (ASM202854v1), Nitrobacter vulgaris DSM10236 (ASM3145399v1), Nitrobacter winogradskyi Nb-255 (ASM1272v1), Nitrobacter winogradskyi Nb-106 (ASM2417141v1), and Nitrobacter winogradskyi NBRC14297 (ASM653954v1), and Nitrobacter sp. JJSN (ASM5191248v1).

[0059] II. Experimental results The nitrifying bacteria Nitrobacter sp.The genome assembly results of TKz-YC01 showed that the strain contained a circular chromosome and a circular large plasmid with lengths of 3938446bp (GC content of 61.62%) and 123343bp (GC content of 59.61%), respectively. The total genome length was 4061789bp and the total GC content was 61.56%.

[0060] Nitrifying bacteria Nitrobacter sp. The average nucleotide consistency results of TKz-YC01 are shown in the figure below. Figure 3 As shown, Nitrifying Bacillus Nitrobacter sp. The average amino acid consistency results of TKz-YC01 are shown in the figure below. Figure 4 As shown, Nitrifying Bacillus Nitrobacter sp. The DNA-DNA hybridization similarity results of TKz-YC01 are shown in Table 2.

[0061] Table 2 Nitrifying Bacteria Nitrobacter sp. DNA-DNA hybridization similarity results of TKz-YC01

[0062] The results showed that: Nitrifying bacteria Nitrobacter sp. TKz-YC01 had ANI, AAI, and DDH values ​​lower than the aforementioned thresholds compared to other Nitrobacter strains, with the highest ANI value being 93.48%, the highest AAI value being 93.12%, and the highest DDH value being 51.9%.

[0063] Therefore, nitrifying bacteria Nitrobacter sp. TKz-YC01 should be identified as a new species of fungus.

[0064] Example 3 Nitrifying Bacillus Nitrobacter sp. Applications of TKz-YC01 I. Under different oxygen concentrations Nitrobacter sp. TKz-YC01 consumption of nitrite 1. Experimental Methods Nitrobacter sp. Preparation of concentrated TKz-YC01 bacterial culture: Take 100 mL of the pure Nitrobacter culture obtained in Example 1, aliquot it into 50 mL centrifuge tubes, centrifuge at 12000×g for 20 min, discard the supernatant and collect the precipitate, resuspend the precipitate with 1×PBS buffer, transfer it to a 2 mL centrifuge tube, centrifuge at 12000×g for 20 min, discard the supernatant and collect precipitate 1, and resuspend the precipitate with 1×PBS buffer to obtain the concentrated TKz-YC01 bacterial culture. Nitrobacter sp. TKz-YC01 concentrated bacterial solution.

[0065] Add 50 mL of 1×AOM medium to a 100 mL capped Erlenmeyer flask, then add sodium nitrite (NaNO2) to bring the nitrite concentration in the flask to 8 mM. Next, add 50 μL of [unspecified ingredient / component]. Nitrobacter sp. The concentrated bacterial culture of TKz-YC01 was then transferred to a shaker at 28℃ and 60 rpm for incubation in the dark. When the nitrite in the conical flask was completely consumed (i.e., the concentration was 0), nitrite was added to restore the concentration to 8 mM. During the incubation process, the nitrite and nitrate concentrations in the conical flask were measured every 24 hours (day). Three replicate conical flasks were set up in parallel, and the mean ± standard deviation was taken as the result.

[0066] Next, the shaking speed was adjusted to 0 rpm, with all other conditions unchanged, and the concentrations of nitrite and nitrate in the conical flask were measured during the culture process, as well as the dissolved oxygen content in the conical flask. The shaking speed was then adjusted to 120 rpm, with all other conditions unchanged, and the concentrations of nitrite and nitrate in the conical flask were measured during the culture process, as well as the dissolved oxygen content in the conical flask. Finally, the shaking speed was adjusted to 180 rpm, with all other conditions unchanged, and the concentrations of nitrite and nitrate in the conical flask were measured during the culture process, as well as the dissolved oxygen content in the conical flask.

[0067] When culturing in shakers at different speeds, take one bottle of each. Nitrobacter sp. The concentrated bacterial culture of TKz-YC01 was cultured in a shaker, and the corresponding dissolved oxygen content (DO) was measured using a dissolved oxygen meter.

[0068] 2. Experimental Results Nitrobacter sp. The results of the TKz-YC01 nitrite consumption test are shown in the figure below. Figure 5 As shown, Figure 5 In this context, A represents the daily nitrite level during the culture process in the conical flasks at various rotation speeds. Figure 5 In the figure, B represents the daily nitrate level during the culture process in the conical flasks at various rotation speeds. Figure 5 In this context, C represents the dissolved oxygen content in the conical flask at each rotational speed.

[0069] The results showed that in the Erlenmeyer flasks with rotation speeds of 60 rpm, 120 rpm, and 180 rpm, the 8 mM nitrite in the flasks was completely consumed by the third day of culture, while the Erlenmeyer flasks in the static group (rotation speed of 0 rpm) still had 1.5 ± 0.3 mM of nitrite remaining. After adding nitrite to 8 mM (on the fourth day), the nitrite consumption rate in the Erlenmeyer flasks with rotation speeds of 60 rpm, 120 rpm, and 180 rpm further accelerated, and the newly added nitrite was almost completely consumed by the fifth day. In contrast, the Erlenmeyer flasks with rotation speeds of 0 rpm had 4.63 ± 0.55 mM of nitrite remaining on the fifth day, and it was not completely consumed until the seventh day of culture.

[0070] The dissolved oxygen (DO) in the conical flask with a rotation speed of 0 rpm was 4.5±0.05 mg / L, the dissolved oxygen (DO) in the conical flask with a rotation speed of 60 rpm was 5.53±0.09 mg / L, the dissolved oxygen (DO) in the conical flask with a rotation speed of 120 rpm was 7.01±0.02 mg / L, and the dissolved oxygen (DO) in the conical flask with a rotation speed of 180 rpm was 7.36±0.10 mg / L; it can be seen that even though there is a significant difference in the dissolved oxygen level in the conical flask at different rotation speeds, the oxidation rate of nitrite by the bacteria is relatively close when the rotation speed reaches 60 rpm or above, indicating that the DO has reached 5.53 mg / L, which meets the growth requirement of the nitrifying bacteria Nitrobacter sp. The growth requirement of TKz-YC01, further improvement of dissolved oxygen will not promote the activity of the strain, and the activity of the strain will not be consistent in a high concentration of DO environment.

[0071] And the oxidation process of nitrite in each conical flask and the production process of nitrate do not completely follow the 1:1 relationship, specifically, for every 16 mM of nitrite consumed, 13.57±0.23 mM of nitrate can be produced, with a conversion efficiency of 84.81%.

[0072] As shown above, the nitrifying bacteria obtained in Example 1 Nitrobacter sp. TKz-YC01 can effectively consume nitrite ions and convert them into nitrate ions, which can be used to degrade nitrite in the environment (especially in water environment).

[0073] II. Ammonia nitrogen degradation test 1. Experimental method In a 100 mL capped conical flask, 50 mL of 1×AOM medium was added, sodium nitrite solution was added to make the concentration of nitrite in the capped conical flask 20 mM, ammonium chloride was added to make the concentration of ammonium ion in the capped conical flask 0.05 mM, then 50 μL of Nitrobacter sp. TKz-YC01 concentrated bacteria solution was added to the conical flask, then it was transferred to a 28°C, 180 rpm shaking incubator for dark culture for 7 days, and the concentration of nitrite, nitrate and ammonium in the conical flask was detected every day during the culture process; three replicates were set in parallel.

[0074] The concentration of ammonium ion in the capped conical flask was adjusted to 0.1 mM, and the rest of the conditions remained unchanged for testing.

[0075] 2. Experimental results The experimental results of the ammonia nitrogen degradation test are shown in Figure 6 A is the nitrite concentration result graph in the ammonia nitrogen degradation test, Figure 6 Figure 6 ​In the graph, B represents the nitrate concentration results from the ammonia nitrogen degradation test. Figure 6 In the figure, C represents the ammonium concentration results from the ammonia nitrogen degradation test experiment.

[0076] The results showed that during days 0-3 of the culture process, the consumption of nitrite, ammonium salt, and nitrate production occurred simultaneously in both the 0.05 mM and 0.1 mM ammonium flasks. On day 3, the initial nitrite concentration of 20 mM in the flasks was completely depleted, and 17.28 ± 0.19 mM nitrate was accumulated. On day 3, ammonium chloride in the 0.05 mM ammonium flask was completely depleted, while 0.014 ± 0.004 mM ammonium salt remained in the 0.1 mM ammonium flask. After the nitrite was completely depleted, the ammonium salt concentration in the flasks remained relatively stable and did not decrease further.

[0077] Results: The nitrifying bacteria obtained in Example 1... Nitrobacter sp. TKz-YC01 can utilize ammonium salts as a nitrogen source through the ammonium assimilation pathway. For every 20 mM of nitrite it metabolizes, it assimilates approximately 0.086 mM of ammonium salt, indicating that this strain also possesses excellent ammonia nitrogen degradation capabilities.

[0078] III. Cyanate Degradation Test 1. Experimental Methods Experimental group: 50 mL of 1×AOM medium was added to a 100 mL Erlenmeyer flask with a cap. Sodium nitrite was added to bring the nitrite concentration in the flask to 16 mM. Sodium cyanate was then added to bring the cyanate concentration in the flask to 0.1 mM. Finally, 50 μL of [unspecified substance] was added to the flask. Nitrobacter sp. The concentrated bacterial culture of TKz-YC01 was then transferred to a shaker at 28°C and 180 rpm and cultured in the dark for 7 days. During the culture, the concentrations of nitrite, nitrate, ammonium and cyanate in the conical flasks were measured daily. Three replicates were set up in parallel.

[0079] The difference between the control group and the experimental group is: Nitrobacter sp. The concentrated bacterial solution of TKz-YC01 is added after high-temperature sterilization, and the rest of the treatment is exactly the same.

[0080] 2. Experimental Results The experimental results of cyanate degradation test are shown in the figure below. Figure 7 As shown, Figure 7 In the figure, A represents the experimental results of the control group. Figure 7 B in the figure represents the experimental results of the experimental group.

[0081] The results showed that no nitrite consumption or nitrate formation was observed in the control group, indicating that... Nitrobacter sp.Nitrobacter sp. The live bacteria in the concentrated bacterial solution of TKz-YC01 were completely inactivated; while the concentrations of nitrite and nitrate in the experimental group changed significantly with the increase of culture time, and the nitrite was completely depleted by day 6.

[0082] In both the control and experimental groups, the cyanate concentration gradually decreased with increasing culture time. On day 0, the theoretical cyanate concentration in the control group was 0.1 mM, and the measured concentration was 0.098 ± 0.003 mM, with no ammonium salt (ammonium ion) detected. On day 6, the cyanate concentration was 0.056 ± 0.007 mM, with 0.021 ± 0.004 mM of ammonium salt detected. In the experimental group, the cyanate concentration on day 0 was 0.097 ± 0.001 mM, with no ammonium salt detected. On day 6, the cyanate concentration decreased to 0.016 ± 0.004 mM, with a degradation rate of 83.51%, and ammonium salt was also basically undetectable, indicating that the nitrogen element in the initial sodium hydrochloride was effectively degraded.

[0083] In the control group, cyanate underwent chemical self-degradation to generate ammonium salt, resulting in a continuous decrease in the concentration of cyanate ions and a continuous increase in the concentration of ammonium ions in the conical flask. At that time, the sum of the two ion concentrations remained basically stable, indicating that the nitrogen element in cyanate was not degraded.

[0084] Therefore, the nitrifying bacteria obtained in Example 1 Nitrobacter sp. TKz-YC01 can effectively remove cyanide from the environment, achieving effective denitrification.

[0085] IV. Ammonium Salt Tolerance Test 1. Experimental Methods Add 50 mL of 1×AOM medium to a 100 mL Erlenmeyer flask with a cap. Add sodium nitrite to bring the nitrite concentration in the flask to 16 mM. Then add ammonium chloride to bring the ammonium ion concentration in the flask to 10 mM. Finally, add 50 μL of [unspecified ingredient - likely a specific ingredient or solution] to the flask. Figure 8 The concentrated bacterial culture of TKz-YC01 was then transferred to a shaker at 28°C and 180 rpm and cultured in the dark for 7 days. During the culture, the concentrations of nitrite and nitrate in the conical flasks were measured daily. Three replicates were set up in parallel.

[0086] Next, the concentration of ammonium ions was adjusted to 0 mM, 20 mM and 30 mM in sequence, with the other treatments being exactly the same, and the concentrations of nitrite and nitrate were measured during the culture process.

[0087] 2. Experimental Results The results of the ammonium salt tolerance test are shown in the figure below. Figure 8 As shown, Figure 8 In the graph, A represents the nitrite concentration results. Nitrobacter sp. B in the graph represents the nitrate concentration results.

[0088] The results show that the addition of ammonium salt does not inhibit the nitrobacterium obtained in Example 1 from removing nitrite and producing nitrate. Nitrobacter sp. Both nitrite consumption and nitrate production of TKz-YC01 are promoted, which can accelerate the removal rate of nitrite and the production rate of nitrate; even if the ammonium concentration is as high as 30 mM, it cannot inhibit the nitrobacterium obtained in Example 1 from removing nitrite and producing nitrate. Nitrobacter sp. The activity of TKz-YC01.

[0089] The results show that the nitrobacterium obtained in Example 1 can remove nitrite and produce nitrate even if the ammonium concentration is as high as 30 mM. Nitrobacter sp. TKz-YC01 has excellent ammonium salt tolerance and can still maintain excellent nitrite removal and nitrate production ability under the impact of high concentration of ammonium ions.

[0090] V. Nitrite Tolerance Test 1. Experimental method In a 100 mL conical flask with a cap, 50 mL of 1x AOM medium was added, and sodium nitrite was added to make the nitrite concentration in the conical flask with a cap 10 mM, and then 50 μL of TKz-YC01 concentrated bacterial solution was added to the conical flask, and then transferred to a 28°C, 180 rpm shaking bed for dark culture for 7 days, and the nitrate concentration in the conical flask was detected every day during the culture process; three replicates were set in parallel. Figure 9 In a 100 mL conical flask with a cap, 50 mL of 1x AOM medium was added, and sodium nitrite was added to make the nitrite concentration in the conical flask with a cap 10 mM, and then 50 μL of TKz-YC01 concentrated bacterial solution was added to the conical flask, and then transferred to a 28°C, 180 rpm shaking bed for dark culture for 7 days, and the nitrate concentration in the conical flask was detected every day during the culture process; three replicates were set in parallel.

[0091] Then the nitrite concentration in the conical flask with a cap was adjusted to 20 mM, 30 mM, 40 mM and 50 mM in turn, and the rest was the same, and the nitrate concentration during the culture process was determined.

[0092] 2. Experimental results The nitrite tolerance test results are shown in the figure Nitrobacter sp. The results show that during the 0-3.5 day process, the nitrate concentrations in the conical flasks with different nitrite concentrations are highly coincident, and there is no significant difference as the culture time increases.

[0093] The results show that even if the nitrite concentration is as high as 50 mM, it will not affect the nitrate production rate of the strain, which shows that the nitrobacterium obtained in Example 1 can remove nitrite and produce nitrate even if the ammonium concentration is as high as 30 mM. ​ TKz-YC01 has excellent nitrite tolerance, It should be pointed out finally that the above embodiments are only used to explain the technical solutions of the present application but not to limit the protection scope of the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. A nitrobacterium (Nitrobacter) TKz-YC01 resistant to nitrite and ammonia nitrogen, characterized in that, Nitrobacter sp. The nitrifying bacteria (NITROBACTERIUM sp.) Nitrobacter sp. The preservation number of TKz-YC01 is GDMCC No: 65729, which was preserved in Guangdong Microbial Culture Collection Center on March 7, 2025. ​ 2. The nitrifying bacterium of claim 1, wherein the bacterium is a member of the genus Nitrobacter. Nitrobacter sp. ) TKz-YC01 in the treatment of water bodies.

3. Use according to claim 2, characterized in that, The water body treatment is to reduce the content of nitrite in the water body, reduce the content of cyanate in the water body and / or reduce the content of ammonia nitrogen in the water body.

4. The use of a nitrifying bacterium (Nitrobacter) according to claim 1 for the preparation of a product for the degradation of nitrite. Nitrobacter sp. ) TKz-YC01 for the preparation of a product for the degradation of nitrite.

5. The use of a nitrifying bacterium (Nitrobacter) according to claim 1 for the preparation of a product for the degradation of cyanate. Nitrobacter sp. ) TKz-YC01 for the preparation of a product for the degradation of cyanate.

6. The use of the nitrifying bacillus (Nitrobacter) TKz-YC01 in the preparation of a product for degrading ammonia nitrogen. Nitrobacter sp. ) TKz-YC01 in the preparation of a product for degrading ammonia nitrogen.

7. The use of a nitrifying bacterium according to claim 1 for the preparation of a product resistant to nitrite. Nitrobacter sp. ) TKz-YC01 for the preparation of a product resistant to nitrite.

8. Use of the Nitrobacter sp. TKz-YC01 of claim 1 in the preparation of an ammonium salt-resistant product.

9. Use according to any one of claims 2 to 8, characterized in that, The nitrifying bacteria (A. xylosoxidans TKz-YC01) includes at least one of live bacteria, a culture solution, or a culture supernatant of the nitrifying bacteria (A. xylosoxidans TKz-YC01). Nitrobacter sp. ) TKz-YC01 includes at least one of live bacteria, a culture solution, or a culture supernatant of the nitrifying bacteria (A. xylosoxidans TKz-YC01). Nitrobacter sp. ) TKz-YC01 10. A microbial inoculant, characterized in that, A nitrifying bacterium according to claim 1, Nitrobacter sp. Nitrobacter sp. ) TKz-YC01.

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