Rhodococcus pyridinivorans YB076 and application of rhodococcus pyridinivorans YB076 in biological denitrification of sewage
By growing the YB076 strain of pyridine-philus Rhesus pyridine-philus under different oxygen conditions, the problem of removing ammonia nitrogen, nitr nitrogen and nitroso nitrogen in sewage was solved, and efficient biological nitrogen removal effect in sewage was achieved, and microbial strain resources were expanded.
Patent Information
- Application Number
- CN202410019253.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-01-05
- Publication Date
- 2025-07-25
AI Technical Summary
The prior art is difficult to effectively remove ammonia nitrogen, nitr nitrogen and nitroso nitrogen in sewage, and microbial strain resources are insufficient, resulting in low biological nitrogen removal efficiency in sewage.
The YB076 strain of pyridine-philus Rheumatum pyridine-philus was grown under different oxygen conditions, and nitrogen was removed in wastewater by oxidizing ammonia nitrogen, reducing nitronitrogen and nitroso nitrogen.
This strain can effectively remove ammonia nitrogen, nitronitrogen and nitroso nitrogen under high concentrations of ammonia nitrogen, improve the efficiency of sewage nitrogen removal, expand the resources of microbial bacterial species, and has the advantages of simple operation, high efficiency and environmentally harmlessness.
Smart Images
Figure CN120366093A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to a bacterial strain, Rhodococcus pyridinivorans YB076 ( Rhodococcus pyridinivorans YB076) and its application in biological denitrification treatment of nitrogen-containing sewage, belonging to the field of environmental microbial 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, continuously developing new microbial strain resources and improving the nitrogen removal efficiency are important topics in sewage biological denitrification.
[0003] Rhodococcus pyridinivorans YB076 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 Rhodococcus pyridinivorans YB076 ( Rhodococcus pyridinivorans YB076) that can be used for biological denitrification of sewage and its application in biological denitrification of nitrogen-containing sewage.
[0005] A strain of Rhodococcus pyridinivorans YB076 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 NO: M 20231956, and the deposit date is October 20, 2023. The taxonomic name is Rhodococcus pyridinivorans YB076.
[0006] The 16S rRNA gene sequencing results of the YB076 strain are shown in SEQ ID NO: 1.
[0007] The culture method of the strain YB076 is as follows: (1) Activation of the deposited strain. YB076 can grow in LB medium. Pick the deposit of the YB076 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 YB076 colonies grow.
[0008] (2) Strain scale-up culture. YB076 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), 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 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 conditions are 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 conditions are 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 is 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 YB076 has the strongest ammonia nitrogen removal ability under aerobic conditions.
[0018] The strain YB076 can effectively remove nitrate nitrogen under anaerobic, aerobic, and anoxic conditions.
[0019] The strain YB076 can effectively remove nitrite nitrogen under anaerobic, aerobic, and anoxic conditions.
[0020] The strain YB076 can grow under anaerobic, aerobic, and anoxic conditions.
[0021] The strain YB076 can grow well within the pH range of 5 - 9.
[0022] The strain YB076 can tolerate 9% NaCl and achieve significant biomass proliferation.
[0023] The present invention also provides a biological denitrification agent, which contains the bacterial strain Rhodococcus pyridinivorans YB076, classified and named Rhodococcus pyridinivorans YB076, deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China, deposit number CCTCC M 20231956, and deposit date 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 Rhodococcus pyridinivorans YB076 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 biological purification of sewage, 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 YB076.
[0028] Figure 2 The morphology of strain YB076 (scanning electron microscope).
[0029] Figure 3 Phylogenetic analysis of strain YB076.
[0030] Figure 4 The ability of strain YB076 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 YB076 in DM culture medium. 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 Under different oxygen conditions, the nitrogen removal ability of YB076 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 YB076 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 YB076 under different initial pH culture conditions.
[0035] Figure 9 The growth characteristics of YB076 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 regarded as limiting the scope of the present invention.
[0037] Example 1 Isolation and identification of strain YB076 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 YB076.
[0039] The morphological results of the obtained strain YB076 are shown in Figure 1 . The pure culture colonies of this strain are transparent and slightly red in color, circular in shape, with regular edges, smooth and round surfaces, 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, with a diameter of about 0.8 µm.
[0041] After strain YB076 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 obtained strain sequence information is as 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 a phylogenetic tree was constructed ( Figure 3 ). The results showed that the similarity rate of the 16S rDNA sequence of this strain to the Rhodococcus pyridinivorans 16S rDNA sequence in the database was 100%. This strain was identified as Rhodococcus pyridinivorans .
[0043] Example 2 The ability of YB076 to remove ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen YB076 was respectively cultured in DM medium with ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen contents of 413 mg / L, 160 mg / L, and 505 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 27.8% ( Figure 4 A), and the nitrite nitrogen removal rate was 99.7% ( Figure 4 C). Under anaerobic conditions, the nitrate nitrogen removal rate was 98.3% ( Figure 4 B). It can be seen that YB076 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 YB076 under different oxygen conditions YB076 was cultured in DM culture solution, using ammonium chloride, potassium nitrate, and sodium nitrite as nitrogen sources respectively. It was respectively 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. The ability of YB076 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, YB076 can grow using ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen as nitrogen sources respectively. After 119 h, the biomass (OD600) was 5.9 times ( Figure 5 A), 4.3 times ( Figure 5 B), and 7.9 times ( Figure 5 C) of that at the initial stage of culture respectively. It can be seen that when using nitrite nitrogen, nitrate nitrogen, and ammonia nitrogen as nitrogen sources in DM culture solution, YB076 can all obtain good growth activity, the strain grows rapidly, the biomass accumulates greatly, and nitrite nitrogen is the most suitable nitrogen source.
[0046] Under anaerobic conditions, in DM culture medium, when YB076 uses ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen as nitrogen sources respectively, the growth of the strain is inhibited. The biomass does not increase and is 0.9 times ( Figure 5 D), 0.9 times ( Figure 5 E), 1.0 times ( Figure 5 F) of that at the initial culture.
[0047] The nitrogen removal ability of strain YB076 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 372 mg / L, 154 mg / L, and 714 mg / L respectively, the removal rates of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen after 119 hours are 34.7% ( Figure 6 A), 96.3% ( Figure 6 B), 100% ( Figure 6 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 366 mg / L, 154 mg / L, and 715 mg / L, after YB076 is cultured for 120 hours, the removal rates of nitrate nitrogen and nitrite nitrogen are 92% ( Figure 6 E) and 100% ( Figure 6 F) respectively. However, ammonia nitrogen cannot be removed ( Figure 6 D).
[0048] In summary, YB076 can tolerate high concentrations of ammonia nitrogen and can remove 34.7% of 372 mg / L 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 96% 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 YB076 is cultured in LB culture medium and placed under aerobic (cultured on a shaker at 200 rpm), anaerobic (the culture medium is 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 show that in LB culture medium, with an initial cell concentration (OD600) of 0.3, after culturing for 67 hours, under anaerobic, aerobic, and anoxic conditions, the cell concentrations reach 0.74, 2.18, and 0.76 respectively, increasing by 2.4 times ( Figure 7 A), 7.2 times ( Figure 7 B), and 2.5 times ( Figure 7C). It can be seen that this strain can grow under anaerobic, aerobic and anoxic conditions when the nutrient conditions are sufficient, and it grows best under aerobic conditions.
[0051] Example 5 Growth characteristics of the strain under different initial pH conditions The YB076 strain was cultured in LB medium, and different pH values were set to investigate the growth characteristics of the strain under different initial culture pH conditions. The results are shown in Figure 8 .
[0052] YB076 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 3.2 - 4.4. The biomass of the strain in the treatments with initial pH of 6, 7 and 8 is the highest, and the cell concentrations (OD600) are 4.1, 4.4 and 4.2 respectively.
[0053] Example 6 Growth characteristics of the strain under different salt concentration conditions The YB076 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] YB076 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 strain, the biomass of the strain treated with 1%, 3%, 5% and 7% NaCl is 4.8 times, 3.9 times and 2.9 times that of the initial culture respectively. The growth of the strain treated with 9% NaCl is significantly inhibited, and the biomass of the strain cultured for 148 hours has a certain increase, which is 1.6 times that of the initial culture.
Claims
1. A bacterial strain, Rhodococcus pyridinivorans YB076, classified and named Rhodococcus pyridinivorans YB076, is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China, the deposit number CCTCC M20231956, 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 as 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 said biological denitrification 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, characterized in that, The said 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 said nitrogen-containing sewage refers to the use of sewage containing inorganic active nitrogen, including one or several of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen.
7. The application according to claim 6, characterized in that, The process of denitrifying the said 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 Rhodococcus pyridinivorans YB076 described in claim 1 or 2, with the taxonomic name Rhodococcus pyridinivorans YB076, 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 20231956, and the deposit date being October 20, 2023.
9. The biological denitrification agent according to claim 8, characterized in that, Use of the said biological denitrification agent in biological denitrification of nitrogen-containing sewage, and the said nitrogen-containing sewage refers to the use of sewage containing inorganic active nitrogen, including one or several of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen.
10. The biological denitrification agent according to claim 9, wherein, Use of the said strain in removing one or more of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in nitrogen-containing sewage, and the process of denitrifying the said 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.