Acinetobacter indicus YB070 and application thereof in biological denitrification of sewage

Abstract

The invention provides a bacterial strain YB070 (Acinetobacter indicus YB070), and the bacterial strain is separated from activated sludge of a nitrogen-containing wastewater treatment system. The YB070 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 is preferred to an acidic environment. And when the NaCl concentration is greater than 7%, the growth is inhibited. YB070 can tolerate high-concentration ammonia nitrogen and can effectively remove ammonia nitrogen under the aerobic condition, and the removal rate of 367mg / L ammonia nitrogen can reach 34.1%. Nitrate nitrogen and nitrite nitrogen in a water body can be removed under the aerobic condition and the anaerobic condition. The removal rates of nitrate nitrogen and nitrite nitrogen respectively reach 92% and 100%. The strain has a good application prospect in sewage denitrification.

Description

Technical Field

[0001] The present invention relates to a bacterial strain Acinetobacter indicus YB070 ( Acinetobacter indicus YB070) and its application in biological denitrification treatment of nitrogen-containing sewage, belonging to the technical field of environmental microbiology. 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 understanding biological nitrogen removal from sewage.

[0003] Acinetobacter indicus YB070 is a bacterium isolated from the activated sludge of nitrogen-rich sewage. The present invention will clarify the application of this strain in the field of effectively removing reactive nitrogen in water bodies. Summary of the Invention

[0004] The present invention provides an Acinetobacter indicus YB070 ( Acinetobacter indicus YB070) that can be used for biological denitrification of sewage and its application in biological denitrification of nitrogen-containing sewage.

[0005] An Acinetobacter indicus YB070 was deposited at the China Center for Type Culture Collection on October 2023. The deposit address is Wuhan University, Wuhan, China. The deposit number is: CCTCC M 20231954, and the deposit date is October 20, 2023. The taxonomic name is Acinetobacter indicus YB070.

[0006] The 16S rRNA gene sequencing results of the YB070 strain are shown in SEQ ID NO:1.

[0007] The culture method of the YB070 strain is as follows: (1) Activation of the deposited strain. YB070 can grow in LB medium. Pick the deposit of the YB070 strain stored at -80°C and place it on an LB solid plate, and streak it with a sterile inoculation loop. Incubate it in a biochemical incubator at 28°C until the YB070 colonies grow.

[0008] (2) Strain scale-up culture. YB070 can be cultured in DM medium. The composition of DM medium (for 1 L volume) is: 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), or 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 300 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 balanced and fully remove oxygen. Then it is placed in an anaerobic culture tank for sealed culture.

[0017] The strain YB070 has the strongest ammonia nitrogen removal ability under aerobic conditions.

[0018] The strain YB070 can effectively remove nitrate nitrogen under anaerobic, aerobic, and anoxic conditions.

[0019] The strain YB070 can effectively remove nitrite nitrogen under anaerobic, aerobic, and anoxic conditions.

[0020] The strain YB070 can grow under anaerobic, aerobic, and anoxic conditions.

[0021] The strain YB070 can grow well within the pH range of 5 - 9.

[0022] The strain YB070 can tolerate 9% NaCl, and when the NaCl concentration is less than 7%, significant proliferation of biomass can be achieved.

[0023] The present invention also provides a biological denitrification agent, which contains the bacterial strain Acinetobacter indicus YB070, with the taxonomic name Acinetobacter indicus YB070, which is preserved in the China Center for Type Culture Collection, with the preservation address being Wuhan University, Wuhan, China, and the preservation number CCTCC M 20231954, 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 more of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen. Such as the sediment of black and odorous water bodies caused by industrial production, agricultural nitrogen pollution, and the sediment of eutrophic lakes, streams, and ponds caused by human domestic nitrogen emissions. In some specific implementation application processes, it includes the sediment 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 Acinetobacter indicus YB070 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 can 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 The pure culture single colony morphology of strain YB070.

[0028] Figure 2 The morphology of strain YB070 (scanning electron microscope).

[0029] Figure 3 Phylogenetic analysis of strain YB070.

[0030] Figure 4 The ability of strain YB070 to remove ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen. A is the ammonia nitrogen removal ability; B is the nitrate nitrogen removal ability; C is the nitrite nitrogen removal ability.

[0031] Figure 5 Under different oxygen conditions, the biomass characteristics of YB070 in DM culture medium. A is the biomass change of YB072 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, and F is the biomass change of the strain with nitrite nitrogen as the substrate under anaerobic conditions.

[0032] Figure 6 Under different oxygen conditions, the nitrogen removal ability of YB070 in DM culture medium. 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 Under different oxygen conditions, the growth characteristics of strain YB070 in LB culture medium. 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 The growth characteristics of YB070 under different initial pH culture conditions.

[0035] Figure 9 The growth characteristics of YB070 under different salt concentrations. Specific implementation manners

[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 YB070 500 mL of nitrogen-containing wastewater denitrifying activated sludge from the laboratory (the laboratory of the Bioengineering Discipline 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 YB070.

[0039] The morphological results of the obtained strain YB070 are shown in Figure 1 . The pure culture colonies of this strain are yellow, round, with regular edges, not smooth on the surface, slightly dry, relatively large, and flat 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 or short rod-shaped, with a diameter of about 0.8 µm.

[0041] After culturing strain YB070 in LB liquid medium, the bacterial cells were collected, and 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′ and 1492R: 5′-TACGGYTACCTTGTTACGACTT-3′. The amplified product was sequenced and analyzed by Shanghai Sangon Biotech Co., Ltd. The obtained strain sequence information is shown in SEQ ID NO:1.

[0042] The sequencing results were compared with the 16S rRNA gene sequences already existing in the NCBI database to obtain related strains, and a phylogenetic tree was constructed ( 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 Acinetobacter indicus . Acinetobacter indicus .

[0043] Example 2 The ability of YB070 to remove ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen YB070 was cultured separately in DM medium with ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen contents of 451 mg / L, 156 mg / L, and 720 mg / L respectively 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 30.8% ( Figure 4 A), and the nitrite nitrogen removal rate was 97.4% ( Figure 4 C). Under anaerobic conditions, the nitrate nitrogen removal rate was 91.0% ( Figure 4 B). It can be seen that YB070 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 YB070 under different oxygen conditions YB070 was cultured in DM culture medium, using ammonium chloride, potassium nitrate, and sodium nitrite as nitrogen sources respectively. It was cultured separately under aerobic (biological shaker at 200 rpm / min) and 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) conditions. The ability of YB070 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 medium, YB070 can grow using ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen as nitrogen sources respectively. After 120 h, the biomass (OD600) was 5.3 times ( Figure 5 A), 5.4 times (Figure 5 B), 2.6 times ( Figure 5 C). It can be seen that when ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen are used as nitrogen sources in the DM culture medium, YB070 can obtain good growth activity, the strain grows rapidly, the biomass accumulates greatly, and nitrate nitrogen is the most suitable nitrogen source.

[0046] Under anaerobic conditions, in the DM culture medium, when YB070 uses ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen as nitrogen sources respectively, the growth of the strain is inhibited. Only when nitrite nitrogen is used as the nitrogen source, there is a slight increase in biomass. After 120 hours, the biomass is 1.2 times that of the initial culture ( Figure 5 F). When ammonia nitrogen and nitrate nitrogen are used as nitrogen sources, the biomass after 120 hours is 0.9 times ( Figure 5 D) and 1.0 times ( Figure 5 E) of that at the initial culture.

[0047] The nitrogen removal ability of the YB070 strain is shown in Figure 6 . Under aerobic conditions, in the DM culture medium, when the initial concentrations of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen are 367 mg / L, 153 mg / L, and 723 mg / L respectively, the removal rates of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen after 120 hours are 34.1% ( Figure 6 A), 91.9% ( Figure 6 B), and 100% ( Figure 6 C) respectively. Under anaerobic conditions, in the DM culture medium, when the initial concentrations of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in the culture medium are 368 mg / L, 153 mg / L, and 722 mg / L, after YB070 is cultured for 120 hours, the removal rates of nitrate nitrogen and nitrite nitrogen are 52.9% ( Figure 6 E) and 100% ( Figure 6 F) respectively. However, ammonia nitrogen cannot be removed ( Figure 6 D).

[0048] In summary, YB070 can tolerate high concentrations of ammonia nitrogen and can remove 34.1% of 367 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 92% 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 YB070 was cultured in the 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 (cultured statically in the air) conditions respectively. The biomass of the strain is shown in Figure 7 .

[0050] The results showed that in LB medium, with an initial cell concentration (OD600) of 0.4 for YB070, after 67 hours of cultivation, under anaerobic, aerobic, and anoxic conditions, the cell concentrations reached 0.98, 2.21, and 1.22 respectively, showing increases of 2.6-fold ( Figure 7 A), 5.7-fold ( Figure 7 B), and 3.1-fold ( Figure 7 C). It can be seen that this strain can grow under anaerobic, aerobic, and anoxic conditions when the nutritional 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 YB070 strain was cultured in LB medium with different pH values set to investigate the growth characteristics of the strain under different initial culture pH conditions. The results are shown in Figure 8 .

[0052] YB070 can grow in a relatively wide pH range, and good strain biomass 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 2.6 - 4.5. The biomass of the strain treated with an initial pH of 5 is the highest, and the strain concentration (OD600) reaches 4.5, indicating that the strain grows well in a slightly acidic environment.

[0053] Example 6 Growth characteristics of the strain under different salt concentration conditions The YB070 strain was cultured in LB medium containing different sodium chloride concentrations to investigate the growth characteristics of the strain under different salt concentration stresses. The results are shown in Figure 9 .

[0054] YB070 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 strain concentration, the biomass of the strain treated with 1%, 3%, and 5% NaCl is 6.8-fold, 5.2-fold, and 3.8-fold of the biomass at the initial culture respectively. For the treatments with 7% and 9% NaCl, the growth of the strain is significantly inhibited, and the biomass of the strain cultured for 148 hours has a certain increase, which is 3.3-fold and 1.6-fold of that at the initial culture respectively.

Claims

1. A bacterial strain, Acinetobacter indicus YB070, classified and named Acinetobacter indicus YB070, is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China, and the deposit number CCTCC M 20231954, and the deposit date being 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, wherein 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 Acinetobacter indicus YB070 described in claim 1 or 2, with the taxonomic name Acinetobacter indicus YB070, which is deposited at the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China, and the deposit number CCTCC M 20231954, and the deposit date being October 20, 2023.

9. The biological denitrification agent according to claim 8, wherein Use of the biological denitrification agent in biological denitrification of nitrogen-containing sewage, and 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.

10. The biological denitrification agent according to claim 9, characterized in that, Use of the strain in removing one or more of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in nitrogen-containing sewage, and 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.

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