Pseudomonas stutzeri YB072 and application thereof in biological denitrification of sewage

Abstract

The invention provides a bacterial strain YB072 (Pseudomonas stutzeri YB072), and the bacterial strain is separated from activated sludge of a nitrogen-containing wastewater treatment system. The YB072 strain can grow under anaerobic, aerobic and anoxic conditions, the growth under the aerobic condition is the best, and the growth under the anoxic condition is the second. The strain can grow well in a pH range of 5-9, and can tolerate a NaCl concentration of 5%. YB072 can tolerate high-concentration ammonia nitrogen and can effectively remove ammonia nitrogen under the aerobic condition, and the removal rate of 368 mg / L ammonia nitrogen can reach 52%. Nitrate nitrogen and nitrite nitrogen in a water body can be removed under the aerobic condition and the anaerobic condition. And the removal rates of nitrate nitrogen and nitrite nitrogen respectively reach 95% and 100%. The strain has a good application prospect in sewage denitrification.

Description

Technical Field

[0001] The present invention relates to a bacterial strain, Pseudomonas stutzeri YB072 ( Stutzeri Pseudomonas YB072), and its application in biological denitrification treatment of nitrogen-containing sewage, belonging to the field of environmental microbiology technology. Background Art

[0002] Nitrogen pollution is one of the main types of water pollution. Effectively controlling the reactive nitrogen discharged from industrial sewage and domestic pollution into the environment is an important means to control nitrogen pollution. The reactive nitrogen in water mainly exists in four different forms: ammonia nitrogen (NH4 + -N), nitrate nitrogen (NO3 - -N), nitrite nitrogen (NO2 - -N), and organic nitrogen. Although various microorganisms have been developed to reduce the nitrogen content in sewage and make the sewage meet the discharge standards. However, continuously developing new microbial strain resources and improving the nitrogen removal efficiency are important topics in biological nitrogen removal from sewage.

[0003] Pseudomonas stutzeri YB072 is a bacterium isolated from the activated sludge of nitrogen-rich sewage. The present invention will clarify the application of this strain in effectively removing reactive nitrogen in water and other fields. Summary of the Invention

[0004] The present invention provides a Pseudomonas stutzeri YB072 ( Pseudomonas stutzeri YB072) that can be used for biological denitrification of sewage, and its application in biological denitrification of nitrogen-containing sewage.

[0005] A strain of Pseudomonas stutzeri YB072 was deposited at the China Center for Type Culture Collection on October 2023. The deposit address is Wuhan University, Wuhan, China, and the deposit number is: CCTCC M 20231955, and the deposit date is October 20, 2023. The taxonomic name is Stutzeri Pseudomonas YB072.

[0006] The 16S rRNA gene sequencing result of the YB072 strain is shown in SEQ ID NO: 1.

[0007] The culture method of the strain YB072 is as follows: (1) Activation of the deposited strain. YB072 can grow in LB medium. Pick the deposit of the YB072 strain stored at -80°C and place it on an LB solid plate, and streak it with a sterile inoculation loop, then invert it and culture it in a biochemical incubator at 28°C until the YB072 colonies grow.

[0008] (2) Strain scale-up culture. YB072 can be cultured in DM medium. The composition of DM medium (for 1 L volume) is as follows: KNO3 1 g (or NH4Cl 1.2 g, or NaNO2 1.5 g); sodium succinate 16.88 g; Na2HPO4·12H2O 10.55 g; KH2PO4 1.5 g; MgSO4·7H2O 0.1 g; trace elements 2.0 mL; pH 7.2 - 7.5, made up to 1 L with distilled water. Trace elements (for 1 L volume): Na2EDTA 50 g, CaCl2 5.5 g, MnCl2·4H2O 5.06 g, FeSO4 5 g, ZnSO4·7H2O 2.2 g, CuSO4·5H2O 1.57 g, CoCl2·6H2O 1.6 g, 1 L of distilled water; the culture temperature is 28°C, and it can be cultured under aerobic conditions, static conditions (anaerobic conditions), and anaerobic conditions.

[0009] Application of the described strain in biological denitrification of nitrogen-containing sewage.

[0010] Application of the described strain in removing one or more of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in nitrogen-containing sewage.

[0011] Application of the described strain in denitrifying nitrogen-containing wastewater by oxidizing one or more of ammonia nitrogen, reducing nitrate nitrogen, and reducing nitrite nitrogen.

[0012] The concentration of the ammonia nitrogen is within 400 mg / L, preferably within 350 mg / L, and more preferably within 200 mg / L; The concentration of nitrate nitrogen is within 200 mg / L, preferably within 150 mg / L, and more preferably within 120 mg / L; The concentration of nitrite nitrogen is within 700 mg / L, preferably within 500 mg / L, and more preferably within 400 mg / L.

[0013] The denitrification process of the nitrogen-containing sewage is carried out in an aerobic or air-static environment to remove one or more of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen; or to remove one or two of nitrate nitrogen and nitrite nitrogen in an anaerobic environment.

[0014] The aerobic condition is that the dissolved oxygen concentration in the liquid is ≥2.0 mg / L, or the bacterial liquid culture is shaken (200 r / min) using a constant-temperature shaker for culture.

[0015] The anaerobic condition is that the dissolved oxygen concentration in the liquid is 0.2 - 0.5 mg / L, or the bacterial liquid culture is placed in the air and statically cultured.

[0016] The anaerobic condition means that the dissolved oxygen concentration in the liquid is ≤ 0.2 mg / L, or the bacterial liquid culture is placed in an anaerobic glove box to be fully balanced to remove oxygen. Then it is placed in an anaerobic culture tank for sealed culture.

[0017] The strain YB072 has the strongest ammonia nitrogen removal ability under aerobic conditions.

[0018] The strain YB072 can effectively remove nitrate nitrogen under anaerobic, aerobic, and anoxic conditions.

[0019] The strain YB072 can effectively remove nitrite nitrogen under anaerobic, aerobic, and anoxic conditions.

[0020] The strain YB072 can grow under anaerobic, aerobic, and anoxic conditions.

[0021] The strain YB072 can grow well within the pH range of 5 - 9.

[0022] The strain YB072 can tolerate 5% NaCl and achieve significant biomass proliferation.

[0023] The present invention also provides a biological denitrification agent, which contains the bacterial strain Pseudomonas stutzeri YB072, classified and named Stutzeri Pseudomonas YB072, which is preserved in the China Center for Type Culture Collection, with the preservation address being Wuhan University, Wuhan, China, the preservation number CCTCC M 20231955, and the preservation date being October 20, 2023.

[0024] The application of the described biological denitrification agent in biological denitrification of nitrogen-containing sewage, where the nitrogen-containing sewage refers to sewage containing inorganic active nitrogen including one or several of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen. Such as the sediment at the bottom of black and odorous water bodies caused by industrial production, agricultural nitrogen pollution, and the sediment at the bottom of eutrophic lakes, streams, and ponds caused by human domestic nitrogen emissions. In some specific implementation applications, it includes the sediment at the bottom of black and odorous polluted water ponds, eutrophic pond sediment, and polluted stream sediment, etc.

[0025] The beneficial effects of the present invention are: The present invention discloses a strain of Pseudomonas stutzeri YB072 and its application method for biological denitrification in nitrogen-polluted sewage. This strain can tolerate high concentrations of ammonia nitrogen, can grow under different oxygen conditions, and achieve sewage denitrification. The present invention expands the microbial strain resources for purifying sewage by biological methods, improves the sewage denitrification treatment efficiency, and provides new application approaches and ideas for the development of new processes for sewage treatment and water body purification.

[0026] The present invention has the advantages of simple operation, high efficiency, and harmless products to the environment, and has the potential for popularization and application. Description of the Drawings

[0027] Figure 1 Morphology of pure culture single colonies of strain YB072.

[0028] Figure 2 Morphology of strain YB072 (scanning electron microscope).

[0029] Figure 3 Phylogenetic analysis of strain YB072.

[0030] Figure 4 Ability of strain YB072 to remove ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen. A is the ability to remove ammonia nitrogen; B is the ability to remove nitrate nitrogen; C is the ability to remove nitrite nitrogen.

[0031] Figure 5 Biomass characteristics of YB072 in DM culture medium under different oxygen conditions. A is the biomass change of the strain with ammonia nitrogen as the substrate under aerobic conditions; B is the biomass change of the strain with nitrate nitrogen as the substrate under aerobic conditions; C is the biomass change of the strain with nitrite nitrogen as the substrate under aerobic conditions; D is the biomass change of the strain with ammonia nitrogen as the substrate under anaerobic conditions; E is the biomass change of the strain with nitrate nitrogen as the substrate under anaerobic conditions; F is the biomass change of the strain with nitrite nitrogen as the substrate under anaerobic conditions.

[0032] Figure 6 Nitrogen removal ability of YB072 in DM culture medium under different oxygen conditions. A is the ammonia nitrogen removal ability under aerobic conditions; B is the nitrate nitrogen removal ability under aerobic conditions; C is the nitrite nitrogen removal ability under aerobic conditions; D is the ammonia nitrogen removal ability under anaerobic conditions; E is the nitrate nitrogen removal ability under anaerobic conditions; F is the nitrite nitrogen removal ability under anaerobic conditions.

[0033] Figure 7 Growth characteristics of strain YB072 in LB culture medium under different oxygen conditions. A is the growth characteristics of the strain under anaerobic conditions; B is the growth characteristics of the strain under aerobic conditions; C is the growth characteristics of the strain under hypoxic conditions.

[0034] Figure 8 Growth characteristics of YB072 under different initial pH culture conditions.

[0035] Figure 9 Growth characteristics of YB072 under different salt concentrations. Specific implementation mode

[0036] The present invention will be described in detail below in conjunction with specific embodiments. However, those skilled in the art will understand that the following embodiments are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention.

[0037] Example 1 Isolation and identification of strain YB072 500 mL of activated sludge for denitrification treatment of nitrogen-containing wastewater in the laboratory (Biological Engineering Discipline Laboratory of China Three Gorges University) was added to 3.5 L of enrichment medium (the formula of the enrichment medium is: 1.2 g of NH4Cl, 1.5 g of NaNO2, 0.5 g of NaHCO3, 0.5 g of KHCO3, 0.2 g of MgSO4·7H2O, 0.136 g of CaCl2·2H2O, 0.027 g of KH2PO4, 1 mL of trace element 1, 1.25 mL of trace element 2, 1 L of tap water. Trace element 1: 1.25 g of EDTA, 1.25 g of FeSO4, 250 mL of distilled water. Trace element 2: 1.25 g of EDTA, 0.1075 g of ZnSO4·7H2O, 0.06 g of CoCl2·6H2O, 0.2475 g of MnCl2·4H2O, 0.0625 g of CuSO4·5H2O, 0.055 g of NaMoO4·2H2O, 0.0475 g of NiCl2·6H2O, 0.0035 g of H3BO3, 250 mL of distilled water.). All the above substances were placed in a 5 L CSTR for enrichment culture. The temperature was set at 28 °C, the rotation speed was set at 50 rpm, and the pH was maintained between 7.0 - 8.0 for enrichment culture. The hydraulic retention time was 8 days.

[0038] The enriched microbial flora was separated and purified using a separation medium (composition: 0.5 g of NaNO2; 3.0 g of C4H4Na2O4; 3.0 g of MgSO4·7H2O; 4.8 g of K2HPO4; 2.0 g of NaCl; 0.05 g of MnSO4·4H2O; 0.05 g of FeSO4; 20 g of agar; 1 L of distilled water) to obtain YB072.

[0039] The morphological results of the obtained strain YB072 are shown in Figure 1 . The pure culture colonies of this strain are transparent in color, round in shape, regular at the edges, smooth and round on the surface, and raised in the middle.

[0040] The morphology of strain YB0072 was observed using a scanning electron microscope, and the results are as Figure 2 shown. The strain is spherical in shape, uniform in size, with a diameter of about 0.3 µm.

[0041] After strain YB072 was cultured in LB liquid, the bacterial cells were collected. DNA was extracted using a bacterial DNA extraction kit. The 16S rRNA gene of the ribosome was amplified by PCR using primers 27F: 5′-AGAGTTTGATCMTGGCTCAG-3′, 1492R: 5′-TACGGYTACCTTGTTACGACTT-3′. The amplified product was sequenced and analyzed by Shanghai Sangon Biotech Co., Ltd. The sequence information of the obtained strain is as As shown in SEQ ID NO: 1.

[0042] The sequencing results were compared with the 16S rRNA gene sequences existing in the NCBI database to obtain related strains and construct a phylogenetic tree ( Figure 3 ). The results showed that the similarity rate of the 16S rDNA sequence of this strain to the 16S rDNA sequence in the database was 100%. This strain was identified as Pseudomonas stutzeri . Stutzeri Pseudomonas .

[0043] Example 2 The ability of YB072 to remove ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen YB072 was respectively cultured in DM medium with ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen contents of 413 mg / L, 158 mg / L, and 520 mg / L to investigate the ability of the strain to remove different forms of nitrogen. The results are shown in Figure 4 . Under aerobic conditions, the ammonia nitrogen removal rate was 59.4% (as shown in A in Figure 4 ), and the nitrite nitrogen removal rate was 99.7% (as shown in C in Figure 4 ). Under anaerobic conditions, the nitrate nitrogen removal rate was 96.4% (as shown in B in Figure 4 ). It can be seen that YB072 can tolerate high concentrations of ammonia nitrogen and can effectively remove ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in water.

[0044] Example 3 The deamination ability of YB072 under different oxygen conditions YB072 was cultured in DM culture solution, using ammonium chloride, potassium nitrate, and sodium nitrite as nitrogen sources respectively. It was cultured under aerobic (biological shaker at 200 rpm / min) and anaerobic (the culture solution was replaced with gas to anaerobic in an anaerobic glove box and then placed in an anaerobic culture tank for sealed culture) conditions respectively. The ability of YB072 to remove ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen was investigated.

[0045] The growth situation of the strain is shown in Figure 5 . Under aerobic conditions, in DM culture solution, YB072 could grow using ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen as nitrogen sources respectively. After 119 h, the biomass (OD600) was 7.7 times (as shown in A in Figure 5 ), 9.5 times (as shown in B in Figure 5 ), and 4.0 times (as shown in C in Figure 5 ) of that at the beginning of the culture respectively. It can be seen that when nitrate nitrogen and ammonia nitrogen are used as nitrogen sources in DM culture solution, YB072 has better growth activity, grows rapidly, and accumulates a large amount of biomass. Nitrate nitrogen is the most suitable nitrogen source.

[0046] Under anaerobic conditions, in DM culture medium, YB072 uses ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen as nitrogen sources respectively. Only the biomass cultured with nitrate nitrogen and nitrite nitrogen increased, with the growth rates being 2.0 times (such as Figure 5 in E) and 1.2 times (such as Figure 5 in F) of the initial culture, respectively, and the growth rate became smaller compared to aerobic culture. The biomass with ammonia nitrogen as the nitrogen source decreased instead of increasing, being 0.8 times (such as Figure 5 in D) of the initial culture.

[0047] The nitrogen removal ability of strain YB072 is shown in Figure 6 . Under aerobic conditions, in DM culture medium, when the initial concentrations of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen are 368 mg / L, 154 mg / L, and 715 mg / L respectively, the removal rates of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen after 120 hours are 52.1% (such as Figure 6 in A), 95.7% (such as Figure 6 in B), and 100% (such as Figure 6 in C), respectively. Under anaerobic conditions, in DM culture medium, when the initial concentrations of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in the culture medium are 374 mg / L, 154 mg / L, and 714 mg / L, after YB072 is cultured for 120 hours, the removal rates of nitrate nitrogen and nitrite nitrogen are 94.6% (such as Figure 6 in E) and 100% (such as Figure 6 in F), respectively. However, ammonia nitrogen cannot be removed (such as Figure 6 in D).

[0048] In summary, YB072 can tolerate high concentrations of ammonia nitrogen and can remove 52.1% of 368 mg / L of ammonia nitrogen under aerobic conditions. It can remove nitrate nitrogen and nitrite nitrogen in water under both aerobic and anaerobic conditions. The removal rates of nitrate nitrogen and nitrite nitrogen reach 94.6% and 100% respectively. It can complete denitrification and nitrogen removal under aerobic and anaerobic conditions and has high ammonia nitrogen tolerance and ammonia nitrogen removal ability, indicating that this strain has good application prospects in sewage nitrogen removal.

[0049] Example 4 Growth characteristics of the strain under different oxygen conditions YB072 was cultured in LB culture medium and placed under aerobic (cultured on a shaker at 200 rpm), anaerobic (the culture medium was replaced with gas to anaerobic in an anaerobic glove box and then placed in an anaerobic culture tank for sealed culture), and anoxic (static culture in air) conditions respectively. The biomass of the strain is shown in Figure 7 .

[0050] The results showed that in LB culture medium, with the initial cell concentration (OD600) of YB072 being 0.3, after 67 hours of culture, under anaerobic, aerobic, and anoxic conditions, the cell concentrations reached 0.37, 2.22, and 1.5 respectively, increasing by 1.1 times (such asFigure 7 in A), 6.8 times (such as Figure 7 in B) and 4.5 times (such as Figure 7 in C). It can be seen that this strain can grow under anaerobic, aerobic and anoxic conditions when the nutrient conditions are sufficient, with the best growth under aerobic conditions and the second best under anoxic conditions.

[0051] Example 5 Growth characteristics of the strain under different initial pH conditions The strain YB072 was cultured in LB medium with different pH values, and the growth characteristics of the strain under different initial culture pH conditions were investigated. The results are shown in Figure 8 .

[0052] YB072 can grow in a relatively wide pH range, and good biomass of the strain can be obtained when the initial pH of the culture is in the range of 5 - 9. When cultured for 148 hours, the cell concentration (OD600) accumulates between 3.4 and 3.8. The biomass of the strain in the treatments with initial pH of 8 and 9 is the highest, and the cell concentrations (OD600) are 3.6 and 3.9 respectively.

[0053] Example 6 Growth characteristics of the strain under different salt concentration conditions The strain YB072 was cultured in LB medium containing different sodium chloride concentrations, and the growth characteristics of the strain under different salt concentration stresses were investigated. The results are shown in Figure 9 .

[0054] YB072 can tolerate a 5% NaCl concentration and shows a trend of increasing biomass. As the NaCl concentration increases, the growth of the strain slows down and the accumulation of strain biomass becomes slower. Compared with the initial cell concentration of the culture, the biomass of the strain treated with 1%, 3%, and 5% NaCl is 8.7 times, 4.9 times, and 4.2 times that of the initial culture respectively. The growth of the strain treated with 7% and 9% NaCl is significantly inhibited, and the biomass of the strain cultured for 150 hours only increases slightly, which are 1.8 times and 1.3 times that of the initial culture respectively.

Claims

1. A bacterial strain, Pseudomonas stutzeri YB072, classified and named Pseudomonas stutzeri YB072, is deposited in the China Center for Type Culture Collection. The deposit address is Wuhan University, Wuhan, China. The deposit number is CCTCC M 20231955, and the deposit date is October 20, 2023.

2. The strain according to claim 1, characterized in that: The 16S rRNA gene sequence of this strain is shown in SEQ ID NO:

1.

3. Use of the strain according to claim 1 in biological denitrification of nitrogen-containing sewage.

4. The application according to claim 3, characterized in that The biological denitrification mentioned refers to the use of the strain in removing one or more of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in nitrogen-containing sewage.

5. The application according to claim 4, wherein The use is achieved by the strain oxidizing ammonia nitrogen and reducing one or more of nitrate nitrogen and nitrite nitrogen.

6. The application according to claim 3, wherein The nitrogen-containing sewage mentioned refers to the use of sewage containing one or several of inorganic active nitrogen including ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen.

7. The application according to claim 6, characterized in that, The nitrogen removal process of the nitrogen-containing sewage refers to removing one or more of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in an aerobic or air-static environment; or removing one or two of nitrate nitrogen and nitrite nitrogen in an anaerobic environment.

8. A biological denitrification agent, characterized in that, The medicament contains the bacterial strain Pseudomonas stutzeri YB072 described in claim 1 or 2, with the taxonomic name Pseudomonas stutzeri YB072, which is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China, deposit number CCTCC M 20231955, and the deposit date being October 20, 2023.

9. The biological denitrification agent according to claim 8, characterized in that, Use of the biological denitrification agent in biological denitrification of nitrogen-containing sewage, where the nitrogen-containing sewage refers to the use of sewage containing one or several of inorganic active nitrogen including ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen.

10. The biological denitrification agent according to claim 9, wherein Use of the strain in removing one or more of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in nitrogen-containing sewage, where the nitrogen removal process of the nitrogen-containing sewage refers to removing one or more of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in an aerobic or air-static environment; or removing one or two of nitrate nitrogen and nitrite nitrogen in an anaerobic environment.

Images