A nitrite-oxidizing bacterium of the genus Nitrospira and its application

Abstract

The present invention discloses a Nitrospira The invention relates to nitrite oxidizing bacteria and their applications, belonging to the field of microbial and environmental protection technology. The strain is named Nitrospira sp. Z02, with a deposit number of GDMCC No: 66050, and was deposited in Guangdong Provincial Microbial Culture Collection Center on March 25, 2025. The strain is a new species, belongs to chemoautotrophic type, and expands Nitrospira The strains are diverse and can oxidize nitrite under different oxygen concentrations, have good adaptability to oxygen concentrations, retain oxidation activity within the pH range of 5.5 to 8.5, and can tolerate weakly acidic conditions. They can grow on solid plates and have the potential for molecular modification. Therefore, this strain has good application value in the treatment of nitrite-containing wastewater.

Description

Technical Field

[0001] The present invention belongs to the field of microorganism and environmental protection technology, and particularly relates to a Nitrospira The invention relates to nitrite oxidizing bacteria and application thereof. Background Art

[0002] Nitrification is an important process in the biogeochemical nitrogen cycle. Chemoautotrophic nitrite-oxidizing bacteria (NOB) catalyze the second step of nitrification, namely the oxidation of nitrite to nitrate. NOB have been found to be widely present in natural environments such as oceans, rivers, lakes, and soil.

[0003] Biological denitrification has a wide range of applications due to its mild, green, efficient, and secondary pollution-free characteristics. NOB is commonly found in bacterial agent products and is used for environmental remediation in aquariums, aquaculture, ponds, rivers, and lakes. It is also used in denitrification scenarios in wastewater treatment plants (WWTPs).

[0004] Currently known NOB include 7 genera, among which Nitrospira The NOB of the genus has been shown to be the main NOB in WWTP. Nitrospira NOB, represented by the genus NOB, are difficult to culture or uncultured microorganisms. It often takes several years or even longer to obtain pure strains under laboratory conditions. Currently, only a few strains have been reported. Nitrospira Pure strains of NOB were obtained by the dilution-to-extinction method and were unable to grow alone on solid plates, which greatly limited the application potential of NOB.

[0005] Separation from the environment Nitrospira NOB strains, especially those that can grow on solid plates, can enrich the types of nitrite-oxidizing bacteria, increase the number of strains available in biological denitrification scenarios, and lay the foundation for constructing engineered strains through molecular technology, which can expand Nitrospira It has the application potential of NOB and provides microbial resource guarantee for ecological environment governance, resource geochemical cycle, etc. Summary of the Invention

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a plant that can grow on a solid plate. Nitrospira Belongs to nitrite oxidizing bacteria.

[0007] Another object of the present invention is to provide the above Nitrospira Application of nitrite oxidizing bacteria.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] One Nitrospira It belongs to nitrite-oxidizing bacteria, named Nitrospira sp. Z02, with the deposit number GDMCCNo: 66050, and was deposited on March 25, 2025 in the Guangdong Microbial Culture Collection Center, Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong, China.

[0010] The above-mentioned nitrite oxidizing bacteria are Nitrospira A new species of the genus that can grow as single colonies on solid agar plates.

[0011] above Nitrospira A method for cultivating nitrite-oxidizing bacteria comprises the following steps:

[0012] 1) The above Nitrospira The seed liquid of nitrite-oxidizing bacteria is spread on a solid plate containing nitrite, and after inverted cultivation, dot-shaped colonies grow; or,

[0013] 2) The above Nitrospira The seed liquid of nitrite-oxidizing bacteria is inoculated into a liquid culture medium containing nitrite for cultivation.

[0014] In step 1),

[0015] The solid plate is made by using agar powder as a coagulant.

[0016] The added amount of the agar powder is 15-20 g / L.

[0017] The temperature of the inverted culture is 20-35°C, further 30±1°C.

[0018] The inverted culture time is 30 to 90 days, further 45 to 65 days.

[0019] In step 2),

[0020] The inoculation amount of seed liquid is 0.1~1% (v / v).

[0021] The culture temperature is 20-35°C, further 30±1°C.

[0022] The culture time is more than 5 days, and the time taken for the substrate to be consumed or nearly consumed varies depending on the concentration of the substrate nitrite;

[0023] In one embodiment of the present invention, when the concentration of nitrite is 5 mmol / L, the culture is performed for 7 to 10 days, more preferably 7 to 9 days.

[0024] Preferably, the seed liquid is NitrospiraIt is obtained by inoculating a single colony of nitrite-oxidizing bacteria into a liquid culture medium containing nitrite.

[0025] The culture temperature is 20-35°C, further 30±1°C.

[0026] The incubation time varies depending on the concentration of the substrate nitrite, and the time it takes for the substrate to be completely consumed or nearly consumed varies;

[0027] In one embodiment of the present invention, when the concentration of nitrite is 2 mmol / L, the culture is performed for 10 to 16 days; more preferably 10 to 14 days; and even more preferably 10 to 12 days.

[0028] above Nitrospira The application of nitrite oxidizing bacteria in nitrite oxidation.

[0029] The nitrite oxidation is to oxidize nitrite into nitrate.

[0030] The nitrite oxidation can be carried out in the pH range of 5.5 to 8.5; further, it can be carried out in the pH range of 6.0 to 8.0.

[0031] The application comprises the following steps:

[0032] The above Nitrospira Nitrite-oxidizing bacteria grow in an environment containing nitrite.

[0033] The initial nitrite concentration in the environment is 0 to 50 mmol / L and is not 0; further, it is 1 to 20 mmol / L.

[0034] The environment includes at least one of water and soil.

[0035] The growth is performed at an oxygen concentration of 1% to 21%; further, the growth is performed at an oxygen concentration of 5% to 10%.

[0036] The growth temperature is 20-35°C; further 30±1°C.

[0037] described Nitrospira The nitrite-oxidizing bacteria are chemoautotrophic; they have the activity of oxidizing nitrite to produce nitrate at room temperature and can use nitrite as the only energy substance; they can grow on solid plates and have the basis for constructing engineered strains; they can tolerate weakly acidic conditions; they can oxidize nitrite under different oxygen concentrations and have good adaptability to oxygen concentrations.

[0038] The present invention has the following advantages and effects compared to the prior art:

[0039] The strain Nitrospira sp. Z02 provided by the present invention is a new species, which expands Nitrospira The strains of this genus are diverse and diverse. Strain Z02 can biooxidize nitrite at varying oxygen concentrations (1-21%), retains oxidative activity within a pH range of 5.5-8.5, and tolerates weakly acidic conditions. Furthermore, strain Z02 can grow on solid plates, suggesting potential for molecular engineering. Therefore, this strain has promising application in the treatment of nitrite-containing wastewater. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 The phylogenetic tree diagram based on the 16S rDNA of Nitrospira sp. Z02 (the arrows indicate the direction of the two strains Leptospirillum The 16S sequences of the WT mice were used as outer branches, and dots indicated branches with bootstrap values ​​of not less than 95).

[0041] Figure 2 This is the colony morphology of strain Nitrospira sp. Z02 after 55 days of culture on solid plates.

[0042] Figure 3 This is a graph showing the results of testing the oxidative metabolic characteristics of strain Nitrospira sp. Z02 under 1 mmol / L nitrite.

[0043] Figure 4 This is a graph showing the results of testing the oxidative metabolic characteristics of strain Nitrospira sp. Z02 under 5 mmol / L nitrite.

[0044] Figure 5 This is the result of testing the oxidative metabolic characteristics of the strain Nitrospira sp. Z02 under 10 mmol / L nitrite.

[0045] Figure 6 This is the result of testing the oxidative metabolic characteristics of the strain Nitrospira sp. Z02 under 20 mmol / L nitrite.

[0046] Figure 7 This is a graph showing the results of testing the oxidative metabolic characteristics of strain Nitrospira sp. Z02 under 50 mmol / L nitrite.

[0047] Figure 8 This is a graph showing the results of detecting changes in nitrate concentration of strain Nitrospira sp. Z02 under oxygen concentrations of 1%, 5%, 10%, and 21%.

[0048] Figure 9This is the result of detecting the change of nitrite concentration of strain Nitrospira sp. Z02 under oxygen concentrations of 1%, 5%, 10%, and 21%.

[0049] Figure 10 This is the detection result of nitrate concentration change of strain Nitrospira sp. Z02 at pH values ​​of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0.

[0050] Figure 11 This is the detection result of nitrite concentration change of strain Nitrospira sp. Z02 at pH values ​​of 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0. DETAILED DESCRIPTION

[0051] The present invention is described in further detail below with reference to the examples and accompanying drawings, but the present invention is not limited thereto. Experimental methods in the following examples, where specific experimental conditions are not specified, generally followed conventional experimental conditions or those recommended by the manufacturer. Materials and reagents used were commercially available unless otherwise specified.

[0052] Example 1

[0053] Enrichment, isolation, identification and growth on solid plates of nitrite-oxidizing bacteria Nitrospira sp. Z02.

[0054] (1) Enrichment of nitrite-oxidizing bacteria: Leachate samples (0-30 cm) were collected from the Foshan Waste Treatment Plant using a five-point square sampling method. The environmental samples were washed with PBS buffer solution and inoculated into an enrichment screening inorganic culture medium. The culture was kept in the dark at 30°C. The nitrate and nitrite content was measured during the culture. Under these conditions, the culture was repeatedly subcultured to obtain a stable nitrite-oxidizing bacterial population. The components of the enrichment and screening inorganic culture medium are as follows: 1 g / L NaCl, 0.02 g / L CaCl2·2H2O, 0.05 g / L MgSO4·7H2O, 0.14 g / L KH2PO4, 0.7 g / L K2HPO4, 0.17 g / L NaHCO3, 0.069 g / L NaNO2 (about 1 mmol / L), and 1 / 1000 (v / v) trace element solution. The composition of the trace element solution is as follows: 1.5 g / L FeCl2·4H2O, 100 mg / L MnCl·4H2O, 62 mg / L H3BO3, 70 mg / L ZnCl2, 36 mg / LNa2MoO4·2H2O, 17 mg / L CuCl2·2H2O, 24 mg / L NiCl2·6H2O, and 190 mg / L CoCl2·6H2O, the solvent is deionized water.

[0055] (2) Isolation of single colonies: The obtained stable nitrite-oxidizing bacterial flora was diluted with sterile water in multiple ratios and then inoculated into an enrichment screening inorganic solid culture medium (15-20 g / L agar was added to the enrichment screening inorganic culture medium). The culture was allowed to stand at 30°C under normal oxygen (21%) conditions. Colonies with different appearances such as color, edge, and transparency were picked for further separation and purification. Screening was performed through aerobic nitrite oxidation experiments. The specific steps are as follows:

[0056] Aerobic nitrite oxidation test: Inoculate a single colony into a 125-mL serum bottle containing 50 mL of enriched screening inorganic medium. Incubate at 30°C under normal oxygen (21%) conditions. Samples are taken every 24 hours to determine the nitrate and nitrite content. A decrease in nitrite content and an increase in nitrate content are considered to indicate aerobic nitrite oxidation.

[0057] A strain was obtained through screening and named as strain Z02. In the aerobic nitrite oxidation experiment, the nitrite content decreased and the nitrate content increased with the extension of culture time. The specific morphological and physiological and biochemical characteristics are described in Table 1.

[0058] Table 1 Specific morphological and physiological and biochemical characteristics of strain Z02

[0059]

[0060] Note: “+”: positive; “-”: negative. When detecting available metabolic substrates and carbon sources, the culture medium used was basically the same as the enrichment and screening inorganic culture medium, except that the substances in the table were used to replace NaHCO3 or NaNO2 in the enrichment and screening inorganic culture medium, and liquid cultures were all carried out under normal oxygen conditions (21%). The composition of LB (Luria-Bertani) medium was: tryptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, pH adjusted to 6.5-7.0, and the solvent was deionized water. The composition of R2A medium was: tryptone 0.25 g / L, acid hydrolyzed casein 0.5 g / L, yeast extract powder 0.5 g / L, soluble starch 0.5 g / L, KH2PO4: 0.3 g / L, MgSO4: 0.1 g / L, glucose 0.5 g / L, sodium pyruvate 0.3 g / L, peptone 0.25 g / L, pH adjusted to 7.1-7.3, and the solvent was deionized water.

[0061] (3) Identification of 16S rDNA of strain Z02: The 16S rDNA fragment of nitrite-oxidizing bacteria Z02 was amplified by PCR using the bacterial 16S rDNA universal primers 27F and 1492R. The PCR reaction system was as follows: template DNA 1 μL, upstream primer (10 μmol / L) 1 μL, downstream primer (10 μmol / L) 1 μL, 2×EasyTaq ® 12.5 μL of PCR SuperMix was added to 25 μL with ddH₂O. PCR reaction conditions were: 94°C pre-denaturation for 5 min; 35 cycles of 94°C denaturation for 30 s, 54°C annealing for 30 s, and 72°C extension for 90 s; and 72°C extension for 10 min. The amplified PCR product was sent to Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd. for 16S rDNA sequencing. The results are shown below.

[0062] 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′;

[0063] 1492R: 5′-GGTTACCTTGTTACGACTT-3′.

[0064] Sequencing results:

[0065]

[0066] The sequencing results were compared on NCBI, and the results showed that strain Z02 was Nitrospira defluvii The 16S rDNA sequence of strain Z02 was the most similar, with a sequence coverage of 100% and a sequence similarity of 97.74%. Nitrospira The 16S rDNA sequences of the genus and other strains were used together to construct a phylogenetic tree using the maximum-likelihood method. The TIM3+F+I+G4 model was selected and bootstrap was set to perform 1000 repeated sampling tests. The phylogenetic tree results are shown in Figure 2. Figure 1 shown.

[0067] According to the reference (Kook JK, Park SN, Lim YK, et al. Genome-based reclassification of Fusobacterium nucleatum subspecies at the species level[J]. Current microbiology, 2017, 74: 1137-1147.), the 16S rDNA gene sequence similarity of 98.65% can be used as the threshold for distinguishing the two species. Therefore, based on the above 16S rDNA similarity, it can be judged that strain Z02 is taxonomically similar to Nitrospira The existing species in the genus are different and can be determined as Nitrospira The obtained strain was named Nitrospira sp. Z02, with the accession number GDMCC No: 66050. It was deposited on March 25, 2025, at the Guangdong Microbial Culture Collection, Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, Compound 100, Xianlie Middle Road, Guangzhou.

[0068] (4) Growth of strain Nitrospira sp. Z02 on solid plates:

[0069] The bacterial liquid of strain Nitrospira sp. Z02 was spread on the enriched and screened inorganic solid culture medium and cultured at 30℃ for 55 days to produce colonies. The colony morphology of strain Nitrospira sp. Z02 is as follows: Figure 2 shown.

[0070] Example 2

[0071] The results of the oxidative metabolic characteristics test of nitrite-oxidizing bacteria Nitrospira sp. Z02 at different nitrite concentrations are as follows:

[0072] A single colony of isolated, purified, and identified Nitrospira sp. Z02 was inoculated into a 125-mL serum bottle containing 50 mL of enriched screening inorganic medium (composition as described in Example 1, with a NaNO₂ concentration of 0.138 g / L (approximately 2 mmol / L)). The culture was incubated in the dark at 30°C under normal oxygen (21%) conditions for 10 days to obtain a seed solution. The nitrate concentration reached 1.11 mmol / L at the time of use.

[0073] The seed liquid was inoculated at 1% (V / V) into a 125 mL serum bottle containing 50 mL of enriched screening inorganic culture medium (the composition is shown in Example 1, wherein the sodium nitrite concentrations were set to 1, 5, 10, 20, and 50 mmol / L, respectively). The culture was kept static at 30°C in the dark under normal oxygen (21%) conditions, and the concentrations of nitrite and nitrate were measured during the culture.

[0074] Oxidative metabolic characteristics of strain Nitrospira sp. Z02 at different nitrite concentrations Figures 3 to 7 As shown in the figure, it can be seen that the nitrite concentration in the culture medium of the inoculated strain Z02 gradually decreases over time, while the nitrate concentration gradually increases over time. Figure 3 It can be seen that 1 mmol / L nitrite was completely oxidized after 137 h, the average nitrate generation rate was 7.14 μmol / L·h, and the nitrite conversion rate was 100%. Figure 4 It can be seen that 5 mmol / L nitrite was completely oxidized after 185 h, the average nitrate generation rate was 26.70 μmol / L·h, and the nitrite conversion rate was 100%. Figure 5 It can be seen that 10 mmol / L nitrite was completely oxidized after 257 h, the average nitrate generation rate was 38.19 μmol / L·h, and the nitrite conversion rate was 100%. Figure 6 It can be seen that 20 mmol / L nitrite was completely oxidized after 401 h, the average nitrate generation rate was 48.27 μmol / L·h, and the nitrite conversion rate was 100%. Figure 7 It can be seen that the activity of strain Z02 was affected under the condition of 50 mmol / L nitrite, but it was still able to oxidize nitrite to produce nitrate.

[0075] Example 3

[0076] The results of the nitrite oxidation metabolism characteristics test of nitrite oxidizing bacteria Nitrospira sp. Z02 under different oxygen concentrations are as follows:

[0077] A single colony of isolated, purified, and identified Nitrospira sp. Z02 was inoculated into an enriched, screened inorganic culture medium and cultured for 10 days to obtain a seed solution (the specific method was the same as in Example 2). The nitrate concentration reached 1.20 mmol / L during use.

[0078] The seed liquid was inoculated at 1‰ (V / V) into a 225 mL serum bottle containing 50 mL of enriched screening inorganic culture medium (the composition is shown in Example 1, wherein the sodium nitrite concentration is 1 mmol / L). The bottle was sealed with a butyl rubber stopper and an aluminum cap. Nitrogen was pumped in and the corresponding oxygen was added to obtain a system with a headspace oxygen gas volume fraction of 1%, 5%, 10%, and 21%. The culture temperature was 30°C and the culture was kept in the dark. During the culture, the concentrations of nitrite and nitrate were measured.

[0079] The results of nitrite oxidation metabolism characteristics of strain Nitrospira sp. Z02 under different oxygen concentrations are shown in the figure below. Figures 8 and 9 As shown in the figure, the nitrate concentration in the culture medium inoculated with strain Z02 gradually increased over time, while the nitrite concentration gradually decreased. The oxidation rates at 5% and 10% oxygen concentrations were similar and faster than those at 21%, with the slowest rate at 1% oxygen. Under all four different oxygen concentrations, strain Z02 was able to completely oxidize 1 mmol / L of nitrite within 210 hours, achieving a nitrite conversion rate of 100%.

[0080] These results indicate that strain Z02 has good adaptability to oxygen concentration and can achieve nitrite oxidation under different oxygen concentrations.

[0081] Example 4

[0082] The results of the nitrite oxidation metabolism characteristics test of nitrite oxidizing bacteria Nitrospira sp. Z02 at different pH values ​​are as follows:

[0083] A single colony of Nitrospira sp. Z02 that had been isolated, purified, and identified was inoculated into an enriched, screened inorganic culture medium and cultured for 12 days to obtain a seed solution (the specific method was the same as in Example 2). The nitrate concentration reached 1.92 mmol / L during use.

[0084] The seed solution was inoculated at 1‰ (v / v) into a 125 mL serum bottle containing 50 mL of inorganic medium. 5 mM 2-morpholinoethanesulfonic acid (MES), 4-hydroxyethylpiperazineethanesulfonic acid (HEPES), or tris(hydroxymethyl)aminomethane (TRIS) was added as a pH buffer. The pH was adjusted to 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, and 9.0 using NaOH and HCl. MES was used as a pH buffer at pH 5.0, 5.5, 6.0, and 6.5; HEPES was used at pH 7.0 and 7.5; and TRIS was used at pH 8.0, 8.5, and 9.0. The cultures were incubated in the dark at 30°C under normal oxygen (21%) conditions. Nitrite and nitrate concentrations were measured during the incubation period. The components of the inorganic culture medium are: 1 g / L NaCl, 0.5 g / L KCl, 0.02 g / L CaCl2·2H2O, 0.05 g / L MgSO4·7H2O, 0.01 g / L KH2PO4, 0.042 g / L NaHCO3, 0.069 g / L NaNO2 (approximately 1 mmol / L), and 1 / 1000 (v / v) trace element solution. The composition of the trace element solution is as follows: 1.5 g / L FeCl2·4H2O, 100 mg / L MnCl·4H2O, 62 mg / L H3BO3, 70 mg / L ZnCl2, 36 mg / LNa2MoO4·2H2O, 17 mg / L CuCl2·2H2O, 24 mg / L NiCl2·6H2O, and 190 mg / L CoCl2·6H2O. The solvent is deionized water.

[0085] Oxidative metabolic characteristics of strain Nitrospira sp. Z02 at different pH values Figures 10 and 11 As shown: As can be seen from the figure, the nitrite oxidation rates at pH 6.5, 7.0, and 7.5 are close, slightly higher than the oxidation rates at pH 6.0 and 8.0; its nitrite oxidation activity is partially inhibited at pH 5.5 and 8.5, but its activity is still retained at pH 5.5 (calculated based on the average oxidation rate relative to pH 7.0 within 168 h); at pH 5.0 and 9.0, there is no nitrite oxidation activity.

[0086] These results indicate that strain Z02 can adapt to the pH range of 6.0 to 8.0 and can tolerate weakly acidic conditions.

[0087] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. One plant Nitrospira Belongs to nitrite-oxidizing bacteria, characterized by, described Nitrospira The name of the nitrite-oxidizing bacteria is Nitrospira sp. Z02, the deposit number is GDMCC No: 66050, and it was deposited on March 25, 2025, at the Guangdong Microbial Culture Collection Center, Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong, China.

2. The method according to claim 1 Nitrospira A method for cultivating nitrite-oxidizing bacteria, characterized in that: The steps include: 1) The claim 1 Nitrospira The seed liquid of nitrite-oxidizing bacteria is spread on a solid plate containing nitrite, and after inverted cultivation, dot-shaped colonies grow; or, 2) The claim 1 Nitrospira The seed liquid of nitrite-oxidizing bacteria is inoculated into a liquid culture medium containing nitrite for cultivation.

3. The culture method according to claim 2, wherein: In step 1), the solid plate is made using agar powder as a coagulant; The amount of agar powder added is 15-20 g / L; In step 1), the temperature of the inverted culture is 20-35°C; In step 1), the inverted culture time is 30 to 90 days; In step 2), the culture temperature is 20-35°C.

4. The method according to claim 1 Nitrospira The application of nitrite oxidizing bacteria in nitrite oxidation.

5. The use according to claim 4, characterized in that: The nitrite oxidation is to oxidize nitrite into nitrate.

6. The use according to claim 4, characterized in that: The nitrite oxidation can be carried out in the pH range of 5.5 to 8.

5.

7. The use according to any one of claims 4 to 6, characterized in that: The steps include: The claim 1 Nitrospira Nitrite-oxidizing bacteria grow in an environment containing nitrite.

8. The use according to claim 7, characterized in that: The environment includes at least one of water and soil.

9. The use according to claim 7, characterized in that: The initial nitrite concentration in the environment is 0 to 50 mmol / L and is not 0.

10. The use according to claim 7, characterized in that: The growth is carried out at an oxygen concentration of 1% to 21%; The growth temperature is 20-35°C.

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