A nitrobacterium and its use for water purification

Abstract

The present application belongs to the field of bioengineering technology. Specifically, it relates to a kind of nitrobacter and its use for water purification. The nitrobacter SZG-NOB-003 described in the present application does not need to add additional carbon source, has high affinity for nitrite, and has multiple environmental and antibiotic resistance, and can stably play a purifying role in harsh or complex environment to improve water quality.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology. Specifically, it relates to a nitrifying bacillus and its use in water purification. Background Technology

[0002] Nitrite (NO2) - Nitrite, an intermediate product of ammonia nitrogen oxidation, is widely accumulated in aquaculture systems, and its toxicity can be more than 50 times that of ammonia nitrogen. Even low-concentration exposure (>0.2 mg / L) can lead to hemocyanin deoxygenation, decreased immunity, and large-scale mortality in aquatic animals. Especially in high-density aquaculture, the continuous decomposition of uneaten feed and feces causes nitrite concentrations to repeatedly exceed standards. Traditional water exchange methods are not only costly but also difficult to eradicate the problem due to limited water resources.

[0003] Nitrifying bacteria (NOB) are key microorganisms in the global nitrogen cycle, primarily functioning to oxidize nitrite to nitrate. In natural environments and artificial ecosystems such as wastewater treatment and aquaculture, NOB plays a crucial role in maintaining nitrogen balance and reducing nitrite toxicity. However, known NOBs have some limitations in practical applications. Firstly, most NOBs do not have a high enough affinity for nitrite, resulting in low oxidation efficiency in environments with low nitrite concentrations, failing to effectively control nitrite accumulation. Secondly, natural environments and artificial ecosystems are often complex and variable, subject to various environmental pressures such as salinity changes. Furthermore, intensive aquaculture's long-term reliance on antibiotics (such as quinolones and tetracyclines) leads to the accumulation of large amounts of aerobic oxidases (ARGs) in aquaculture water, sediment, and organisms.

[0004] Existing NOB strains lack sufficient resistance to these environmental stresses and antibiotics, making it difficult to function stably in harsh or complex environments. Therefore, developing a nitrifying bacterium with high nitrite affinity and multiple environmental and antibiotic resistances is of significant practical importance. Summary of the Invention

[0005] In view of this, in a first aspect, the present invention provides a Nitrifying Bacillus (Nitrobacter Winogradskyi) SZG-NOB-003, the accession number of which is CCTCC M 20251405.

[0006] The Nitrifying Bacillus SZG-NOB-003 described in this invention was deposited at the China Center for Type Culture Collection (CCTCC) on June 16, 2025, with accession number CCTCC M 20251405.

[0007] The nitrifying bacteria SZG-NOB-003 described in this invention was screened and isolated from seawater and sediments in Ningbo during winter.

[0008] The nitrifying bacteria SZG-NOB-003 described in this invention does not require the addition of an additional carbon source, has a high affinity for nitrite, and exhibits multiple environmental and antibiotic resistances, enabling it to stably perform its purification function and improve water quality in harsh or complex environments.

[0009] In a second aspect, the present invention provides a biological agent comprising Nitrifying Bacillus SZG-NOB-003 and / or its metabolic enzyme products as described above.

[0010] Thirdly, the present invention provides a water purification treatment method, comprising: applying the nitrifying bacteria SZG-NOB-003 and / or its metabolic enzyme products, or the biological agent described in the present invention, to the water body.

[0011] Furthermore, the method further includes adding 0.01% to 0.05% (by mass) of nitrifying bacteria SZG-NOB-003 solution to the water to be purified. For example, 0.02%, 0.03%, and 0.04% nitrifying bacteria SZG-NOB-003 solution.

[0012] Furthermore, the bacterial culture is added at a rate of approximately 100–5000 mg NO2-N / L / h. Optionally, it is 1000 mg NO2-N / L / h.

[0013] Furthermore, the purification of the water body involves reducing the nitrite content in the water body.

[0014] Furthermore, the water body is an aquarium or aquaculture water body.

[0015] Further, the nitrite content in the water body is greater than or equal to 0.2 mg / L. Further, the nitrite content in the water body is less than or equal to 1 g / L. Optionally, the nitrite content in the water body is less than or equal to 100 mg / L; alternatively, the nitrite content in the water body is less than or equal to 10 mg / L.

[0016] Furthermore, the salinity range of the water body is 0 to 40,000, preferably 0 to 30,000, and more preferably 10,000 to 30,000.

[0017] Furthermore, the water body is rich in antibiotics. Even further, the concentration of the antibiotics is one or more of the following:

[0018] 1) Gentamicin sulfate at concentrations of 0–200 mg / L;

[0019] 2) Tetracycline hydrochloride at concentrations of 0–50 mg / L; or

[0020] 3) Penicillin at concentrations of 0–300 mg / L.

[0021] Furthermore, the concentration of the antibiotic is one or more of the following:

[0022] 1) Gentamicin sulfate at concentrations of 0–100 mg / L;

[0023] 2) Tetracycline hydrochloride at concentrations of 0–20 mg / L; or

[0024] 3) Penicillin at concentrations of 0–100 mg / L.

[0025] Fourthly, the present invention provides the use of the nitrifying bacillus SZG-NOB-003 and / or its metabolic enzyme products described herein for purifying water bodies.

[0026] Furthermore, the purification of the water body involves reducing the nitrite content in the water body.

[0027] Furthermore, the water body is an aquarium or aquaculture water body.

[0028] Furthermore, the nitrite content in the water body is greater than or equal to 0.2 mg / L. Furthermore, the nitrite content in the water body is less than or equal to 10 mg / L.

[0029] Furthermore, the salinity range of the water body is 0 to 40,000, preferably 0 to 30,000, and more preferably 10,000 to 30,000.

[0030] Furthermore, the water body is rich in antibiotics. Even further, the concentration of the antibiotics is one or more of the following:

[0031] 1) Gentamicin sulfate at concentrations of 0–200 mg / L;

[0032] 2) Tetracycline hydrochloride at concentrations of 0–50 mg / L; or

[0033] 3) Penicillin at concentrations of 0–300 mg / L.

[0034] Furthermore, the concentration of the antibiotic is one or more of the following:

[0035] 1) Gentamicin sulfate at concentrations of 0–100 mg / L;

[0036] 2) Tetracycline hydrochloride at concentrations of 0–20 mg / L; or

[0037] 3) Penicillin at concentrations of 0–100 mg / L. Attached Figure Description

[0038] Figure 1Staining observation of bacterial cell morphology for strain SZG-NOB-003.

[0039] Figure 2 This is a scanning electron microscope image of strain SZG-NOB-003.

[0040] Figure 3 Phylogenetic tree of strain SZG-NOB-003 16S rRNA.

[0041] Figure 4 Phylogenetic tree of enzymes in strain SZG-NOB-003NOB-003NxrB.

[0042] Figure 5 The equation diagram for strain SZG-NOB-003 Michaelis-Menten.

[0043] Figure 6 Image of strain SZG-NOB-003 Lineweaver-Burk.

[0044] Figure 7 The effect of different salinities on the degradation performance of strain SZG-NOB-003.

[0045] Figure 8 The effect of gentamicin sulfate on the degradation performance of strain SZG-NOB-003.

[0046] Figure 9 The effect of tetracycline hydrochloride on the degradation performance of strain SZG-NOB-003.

[0047] Figure 10 The image shows the degradation effect of strain SZG-NOB-003 in a freshwater aquarium.

[0048] Figure 11 This is a diagram illustrating the effect of a recirculating aquaculture system using strain SZG-NOB-003. Detailed Implementation

[0049] The present invention will be described in detail below with reference to specific implementation schemes and embodiments, thereby making the advantages and various effects of the present invention more clearly apparent. Those skilled in the art should understand that these specific implementation schemes and embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0050] Unless otherwise specified, the terms used in this application have the common meanings as commonly understood by those skilled in the art.

[0051] The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit this disclosure. When used herein, the singular forms “a,” “an,” and “the” are also intended to include the plural forms unless the context clearly indicates otherwise. It should also be understood that the terms “comprise” and / or “comprising,” when used in this specification, identify the presence of the stated features, integers, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups. When used herein, the term “and / or” includes any and all combinations of the associated listed items.

[0052] In this description, it should be noted that, unless otherwise stated, "above" and "below" include the stated number, and "multiple" in "one or more" means two or more.

[0053] Example 1: Enrichment and Isolation of Nitrifying Bacillus of the Present Invention

[0054] Enrichment of strains

[0055] Enrichment was achieved using winter seawater and sediment from Ningbo. After centrifugation, the supernatant was discarded, and the precipitate was added to the enrichment medium at 10% (w / v). Enrichment was carried out at 28-30℃ and 200 rpm using a shaker. Changes in nitrite nitrogen (NO2-N) and nitrate nitrogen (NO3-N) levels in the medium were monitored daily. Once nitrite nitrogen levels dropped to 0 mg / L, nitrite nitrogen was added back. When nitrate nitrogen levels exceeded 1000 mg / L, the medium was re-inoculated at 10% with a higher nitrite nitrogen content. After two months of acclimatization using this method, the microbial NO2-N degradation efficiency in the enriched solution increased to 100 mg / L·h, yielding an enriched solution with a nitrifying bacteria abundance of 30%. This enriched solution was then purified using a gradient dilution method and a solid dilution plating combined with streak plating.

[0056] Enrichment medium formulation:

[0057] NaNO2 2g / L, NaHCO3 1.86g / L, Na2CO3 0.2g / L, NaCI 0.2g / L, KH2PO4 0.1g / L, MgSO4·7H2O 0.1g / L and FeSO4·7H2O 0.01g / L.

[0058] Isolation and purification of strains

[0059] Dilution coating method and streak plating purification: Dilute the above enriched solution with pure water to a concentration of 10. -1 ~10 -7Seven cell / mL gradients were prepared, with 100 μl of each gradient spread onto solid purification medium. Each gradient was replicated in triplicate. The plates were incubated at 30°C for two weeks, and different colonies that grew on the plates were then streaked for purification.

[0060] After three generations of streak plating, a single strain was finally purified and named SZG-NOB-003.

[0061] Solid culture medium: Add 1.5% to 2% (w / v) agar powder to the enriched culture medium.

[0062] Example 2: Molecular biological identification of the Nitrifying Bacillus SZG-NOB-003 strain of the present invention

[0063] morphology of strain SZG-NOB-003

[0064] Strawberry strain SZG-NOB-003 was streaked onto solid purification medium and incubated at 30°C under aerobic conditions for 2 weeks. SZG-NOB-003 colonies were 0.1–0.5 mm in diameter, transparent, smooth, and glossy, odorless and tasteless. It stained red with Gram stain, therefore this strain is a Gram-negative bacterium (see...). Figure 1 Scanning electron microscopy revealed that the strain was a bacillus, 1–1.5 μm long and 0.2–0.5 μm wide (see [link to study]). Figure 2 ).

[0065] Molecular biological identification of strain SZG-NOB-003

[0066] The genome of strain SZG-NOB-003 was extracted and 16S rDNA was sequenced. The obtained nucleotide sequence is shown in SEQ ID NO.1. The sequencing results were compared with known sequences in the EZbioclound database for similarity, as shown in the figure. Figure 3 As shown, strain SZG-NOB-003 has the highest sequence homology with Nitrobacter Winogradskyi and was identified as Nitrobacter.

[0067] The genome of SZG-NOB-003 was subjected to next-generation sequencing and analysis. Glimmer3 was used for genome-coding gene prediction. The protein sequences of the predicted genes were aligned with the NR, GENES, KEGG, and GO databases using Blastop assays to obtain annotation information for the proteins encoded by the predicted genes. Genome annotation showed that SZG-NOB-003 possesses two functional enzymes, NxrB, that mediate nitrite redox, named NxrB1 and NxrB2, respectively. The enzyme sequences were compared with known sequences in the NCBI database for similarity. Figure 4As shown, NxrB1, NxrB2 and NxrB2 have the highest homology with NxrB in the Nitrobacter sequence.

[0068] In summary, the strain SZG-NOB-003 obtained in this invention is Nitrobacter. Therefore, strain SZG-NOB-003 was deposited on June 16, 2025, at the China Center for Type Culture Collection (CCTCC) (Address: China Center for Type Culture Collection, Wuhan University, No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, Postcode: 430072), with accession number: CCTCC NO: M 20251405, and classified as: Nitrobacter winogradskyi SZG-NOB-003.

[0069] SEQ ID NO.1: >SZG-NOB-003

[0070]

[0071] Example 3: Determination of substrate affinity of the present invention, *Nitrifying Bacillus* SZG-NOB-003

[0072] The strain was cultured to the logarithmic growth phase, centrifuged and washed, and the bacterial suspension concentration was adjusted to OD600nm = 0.5. A 1% inoculation was performed on the bacterial suspension, and the degradation rate of nitrite nitrogen at gradient concentrations of 0.1–10 mg N / L was tested at 28℃ and pH 7.8. The oxidation rate was calculated, and the Km value was obtained using the Michaelis-Menten equation to determine the affinity of the strain for the substrate; a smaller Km value indicates a stronger affinity.

[0073] The substrate affinity of strain SZG-NOB-003 was determined by standardized kinetic tests. The Km value was 0.6 ± 0.24 mg N / L obtained by fitting the Michaelis-Menten equation and the Lineweaver-Burk method. The reaction rate Vmax when the enzyme was saturated with substrate was 714 mg NO2--N / L / h (see...). Figure 5 and 6 This value is significantly lower than the reported Km value of *Nitrobacter winogradskyi* (the Km of *Nitrobacter winogradskyi* is typically 1–5 mg / L, and that of *Nitrobacter vulgaris* is approximately 2–8 mg / L), confirming its high affinity for low concentrations of nitrite. The Vmax is higher than that of most natural nitrifying bacteria (the Vmax of *N. winogradskyi* under standard conditions is approximately 200–500 mg / L / h, while the Vmax of highly efficient engineered strains reported in the literature can reach 600–800 mg / L / h). A high Vmax indicates that this strain has an extremely rapid oxidation rate when the substrate is saturated, making it suitable for treating high-load nitrite pollution (such as industrial wastewater and high-density aquaculture water bodies), and can shorten the treatment cycle or reduce the strain dosage.

[0074] Example 4: Degradation of nitrite nitrogen by the nitrifying bacteria of the present invention under different seawater salinities

[0075] Shake-flask experiments were conducted on strain SZG-NOB-003 in 200 ml of artificial seawater with different total salinity concentrations. The pH was controlled at 7.8±0.2, temperature at 28±1℃, and shaker speed at 200 rpm. The initial nitrite nitrogen concentration was 10 mg / L. After adjusting the initial growth rate to 1000 mg NO2--N / L / h, 0.1 ml of the strain was added to the shake flask at a dosage of 0.05%. Nitrite nitrogen concentration was measured periodically. Specific results are as follows: Figure 7As shown, at salinity levels of 0–3%, strain HZ-N-008 showed almost no inhibition on nitrite degradation efficiency. At salinity levels of 10,000–20,000, the degradation rate reached over 99% within 33 hours, and at 30,000, the degradation rate remained above 99% within 37 hours. At a salinity of 40,000, nitrite degradation was slightly inhibited, requiring 54 hours to degrade 10 mg / L of nitrite. The typical salinity range for seawater aquaculture is 10,000–30,000; therefore, strain SZG-NOB-003 is suitable for both seawater and brackish water aquaculture. In conclusion, the optimal salinity range for strain SZG-NOB-003 is 0–3% (10,000–30,000 salinity).

[0076] Example 5: Degradation of nitrite nitrogen by the present invention using nitrifying bacteria in gentamicin sulfate.

[0077] Antibiotic resistance experiments were conducted on strain SZG-NOB-003 in 200 ml of simulated wastewater. Antibiotic concentrations ranged from 0 to 200 mg / L. The pH was controlled at 7.8 ± 0.2, the shaker speed at 200 rpm, and the initial nitrite nitrogen concentration at 10 mg / L. After adjusting the initial growth rate to 1000 mg NO₂⁻⁻⁶ / L / h, the strain was added to the shake flask at a 1% dosage (2 mL). Nitrite nitrogen concentrations were measured periodically. Specific results are shown below. Figure 8 As shown, gentamicin sulfate exhibits a "low-promote, high-inhibitory" effect on the nitrite nitrogen degradation performance of strain SZG-NOB-003: concentrations below 5 μg / mL have no significant effect on degradation efficiency, and may even enhance substrate uptake efficiency by increasing cell membrane permeability; concentrations above 50 mg / L inhibit ribosomal protein synthesis and cell membrane integrity, leading to a decrease in degradation rate. For example, at 100 mg / L, the degradation rate at 80 minutes is 8.6% lower than the control group, and the complete degradation time is extended from 80 minutes to 100 minutes. However, concentrations below 100 mg / L can still achieve complete degradation within 100 minutes. When the concentration reaches 200 mg / L, a 70% degradation rate can still be achieved in 100 minutes, with nitrite nitrogen residue at 3.897 mg / L. This strain shows strong tolerance to gentamicin (the dosage of gentamicin sulfate used in conventional aquaculture is 4–50 mg / L), making it suitable for aquaculture systems using antibiotic adjuvant therapy and for the biological purification of antibiotic-containing wastewater.

[0078] Example 6: Degradation of nitrite nitrogen by the nitrifying bacteria of the present invention in tetracycline hydrochloride.

[0079] Antibiotic resistance experiments were conducted on strain SZG-NOB-003 in 200 ml of simulated wastewater. Antibiotic concentrations ranged from 0 to 200 mg / L. The pH was controlled at 7.8 ± 0.2, the shaker speed at 200 rpm, and the initial nitrite nitrogen concentration at 10 mg / L. After adjusting the initial growth rate to 1000 mg NO₂⁻⁻⁶ / L / h, the strain was added to the shake flask at a 1% dosage (2 mL). Nitrite nitrogen concentrations were measured periodically. Specific results are shown below. Figure 9 As shown, at low concentrations of tetracycline hydrochloride (5–10 mg / L), the degradation efficiency of this strain was not significantly different from the control group, and it could completely degrade nitrite nitrogen within 80 minutes, indicating its tolerance to low-dose tetracycline, possibly due to an active efflux mechanism or the action of ribosomal protective proteins. Concentrations above 20 mg / L slightly inhibited degradation activity, and at 50 mg / L, the degradation rate was 84.7% after 120 minutes, with only 1.93 mg / L of nitrite nitrogen remaining. This strain exhibits better tolerance to tetracycline than most nitrifying bacteria (conventional nitrifying bacteria lose activity at a tetracycline concentration of 35 mg / L), making it suitable for aquaculture water purification scenarios requiring low-dose tetracycline adjuvant therapy.

[0080] Example 7: Application of the Nitrifying Bacillus of the present invention in freshwater aquariums during winter.

[0081] The water temperature was controlled at 20±1℃, pH at 7.5±0.3, and dissolved oxygen at ≥5mg / L. The initial nitrite nitrogen concentration was 0.5mg / L. The initial inoculum rates of SZG-NOB-003 and commercially available bacterial agents were adjusted to 800mg NO2--N / L / h. Then, 10mL (containing 50mg / L penicillin) was added to both the experimental and control aquariums. A blank control group was also included without any bacterial strains. The nitrite nitrogen concentration was monitored periodically. After two consecutive days of addition, the nitrite nitrogen concentration in the aquarium water reached zero. Specific results are shown below. Figure 10 As shown, strain SZG-NOB-003, when applied to freshwater aquariums, can rapidly reduce the concentration of nitrite nitrogen in the aquarium, and the effect is long-lasting, maintaining it within the safe threshold of 0.1–0.2 mg / L for 168 hours. In contrast, existing nitrifying bacteria, due to their low affinity, can only reduce the concentration of nitrite nitrogen to around 0.5 mg / L.

[0082] Example 8: Application of the Nitrifying Bacillus of the present invention in summer seawater recirculating aquaculture.

[0083] An experiment was conducted using a 100m³ recirculating aquaculture system for Litopenaeus vannamei shrimp in a certain region. 3The average daily feed intake in the aquaculture pond system resulted in a nitrite nitrogen content of 0.2-0.5 mg / L in the influent to the biological treatment pond, with a pH of 8.5-8.6 and a salinity of 20,000. Strain SZG-NOB-003 was inoculated at a ratio of 0.05%, and changes in nitrite nitrogen content in the aquaculture pond were continuously monitored. After 3 days of operation, the nitrite nitrogen content stabilized between 0 and 0.004 mg / L. Specific indicators are as follows... Figure 11 As shown, when strain SZG-NOB-003 is applied to marine aquaculture, it can rapidly reduce the concentration of nitrite nitrogen in the water, and the effect is long-lasting, maintaining it within the safe threshold of 0.1-0.2 mg / L for 168 hours.

Claims

1. A type of nitrifying bacillus ( Nitrobacter Winogradskyi SZG-NOB-003, characterized in that, Its accession number is CCTCC M 20251405.

2. A water purification method, comprising: The nitrifying bacteria SZG-NOB-003 described in claim 1 is applied to water.

3. The method according to claim 2, characterized in that, The method further includes adding 0.01% to 0.05% of nitrifying bacteria SZG-NOB-003 solution to the water body to be purified.

4. The method according to claim 2 or 3, characterized in that, The purpose of purifying the water is to reduce the nitrite content in the water.

5. The method according to claim 2 or 3, characterized in that, The water body in question is an aquarium or aquaculture water body.

6. The use of the Nitrifying Bacillus SZG-NOB-003 as described in claim 1 for purifying water bodies.

7. The use according to claim 6, characterized in that, The purpose of purifying the water is to reduce the nitrite content in the water.

8. The use according to claim 6 or 7, characterized in that, The water body in question is an aquarium or aquaculture water body.

9. The use according to claim 6 or 7, characterized in that, The water body meets at least one of the following conditions: The nitrite content in the water body is greater than or equal to 0.2 mg / L; The nitrite content in the water body is less than or equal to 1 g / L; The salinity range of the water body is 0 to 40,000; or The water body in question is rich in antibiotics.

10. The use according to claim 9, characterized in that, The salinity of the water body ranges from 0 to 30,000.

11. The use according to claim 10, characterized in that, The salinity of the water body ranges from 10,000 to 30,000.

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