Fragrance-removing alcaligenes YB082 and application of fragrance-removing alcaligenes YB082 in biological nitrogen removal of sewage
By using the YB082 strain of descentiformis YB082 to treat sewage under different oxygen conditions, the problem of removing multiple active nitrogen in sewage was solved, efficient sewage nitrogen removal effect was achieved, microbial bacterial strain resources were expanded, and sewage treatment efficiency was improved.
Patent Information
- Application Number
- CN202410019248.6
- 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 various forms of active nitrogen in sewage treatment, especially high concentrations of ammonia, nitronitrogen and nitroso nitrogen, and the resources of microbial strains are limited, resulting in low denitrification efficiency of sewage.
The YB082 strain of degassinaceous Bacillus was grown under different oxygen conditions, and the removal of various active nitrogens in the wastewater was achieved through ammonia oxidation, reduction of nitr nitrogen and nitrosin nitrogen.
This strain can grow under high concentrations of ammonia nitrogen, effectively remove ammonia nitrogen, nitrogen and nitroso nitrogen, improve the efficiency of sewage denitrogenation, expand the resources of microbial bacterial species, and provide new sewage treatment methods.
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Abstract
Description
Technical Field
[0001] The present invention relates to an alkalescent bacterium strain, Alcaligenes odorifaciens YB082 ( Alcaligenes defragrans YB082) 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 active nitrogen discharged from industrial sewage and domestic pollution into the environment is an important means to control nitrogen pollution. The active 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 the biological denitrification of sewage.
[0003] Alcaligenes odorifaciens YB082 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 active nitrogen in water and other fields. Summary of the Invention
[0004] The present invention provides an Alcaligenes odorifaciens YB082 ( Alcaligenes defragrans YB082) that can be used for biological denitrification of sewage, and its application in biological denitrification of nitrogen-containing sewage.
[0005] An Alcaligenes odorifaciens YB082 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 20231957, and the deposit date is October 20, 2023. The taxonomic name is Alcaligenes defragrans YB082.
[0006] The 16S rRNA gene sequencing result of the YB082 strain is shown in SEQ ID NO:1.
[0007] The culture method of the strain YB082 is as follows: (1) Activation of the deposited strain. YB082 can grow in LB medium. Pick the deposit of the YB082 strain stored at -80°C and place it on an LB solid plate, and streak it with a sterile inoculation loop, then place it upside down in a biochemical incubator at 28°C for cultivation until the YB082 colonies grow.
[0008] (2) Strain scale-up culture. YB082 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 450 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 180 mg / L, and more preferably within 150 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 described 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 cultured statically.
[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 YB082 has the strongest ammonia nitrogen removal ability under aerobic conditions.
[0018] The strain YB082 can effectively remove nitrate nitrogen under anaerobic, aerobic, and anoxic conditions.
[0019] The strain YB082 can effectively remove nitrite nitrogen under anaerobic, aerobic, and anoxic conditions.
[0020] The strain YB082 can grow under anaerobic, aerobic, and anoxic conditions.
[0021] The strain YB082 can grow well within the pH range of 5-9.
[0022] The strain YB082 can tolerate 7% NaCl and achieve biomass proliferation.
[0023] The present invention also provides a biological denitrification agent, which contains the bacterial strain Alcaligenes odorifaciens YB082, classified and named Alcaligenes defragrans YB082, deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China, the deposit number CCTCC M 20231957, and the deposit date being October 20, 2023.
[0024] The application of the described biological denitrification agent in biological denitrification of nitrogen-containing sewage. 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 bottom mud of black and odorous water bodies caused by industrial production, agricultural nitrogen pollution, and the bottom mud of eutrophic lakes, streams, and ponds caused by human domestic nitrogen emissions. In some specific implementation application processes, it includes the bottom mud of black and odorous polluted water ponds, eutrophic pond bottom mud, polluted stream bottom mud, etc.
[0025] The beneficial effects of the present invention are: The present invention discloses a strain of Alcaligenes odorifaciens YB082 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 and improves the sewage denitrification treatment efficiency. It 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 YB082.
[0028] Figure 2 Morphology of strain YB082 (scanning electron microscope).
[0029] Figure 3 Phylogenetic analysis of strain YB082.
[0030] Figure 4 Ability of strain YB082 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 strain YB082 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 strain YB082 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 YB082 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 YB082 under different initial pH culture conditions.
[0035] Figure 9 Growth characteristics of YB082 under different salt concentrations. Detailed 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 YB082 500 mL of activated sludge for denitrification treatment of nitrogen-containing wastewater from 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 of 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 and 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 YB082.
[0039] The morphological results of the obtained strain YB082 are shown in Figure 1 . The pure culture colonies of this strain were yellowish-white, round in shape, with irregular edges and sunken surfaces.
[0040] The morphology of strain YB0082 was observed using a scanning electron microscope, and the results are as Figure 2 shown. The strain was rod-shaped, with uniform thickness and a length of about 1.5 µm.
[0041] After strain YB082 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′ and 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 shown as SEQ ID NO:1.
[0042] The sequencing results were aligned 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 with the Alcaligenes defragrans 16S rDNA sequence in the database was 99.86%. This strain was identified as Alcaligenes defragrans .
[0043] Example 2 The abilities of YB082 to remove ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen YB082 was respectively cultured in DM medium with ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen contents of 423 mg / L, 153 mg / L, and 526 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 36.9% ( Figure 4 A), and the nitrite nitrogen removal rate was 99.6% ( Figure 4 C). Under anaerobic conditions, the nitrate nitrogen removal rate was 97.1% ( Figure 4 B). It can be seen that YB082 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 YB082 under different oxygen conditions YB082 was cultured in DM culture medium, 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 medium 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 abilities of YB082 to remove ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen were investigated.
[0045] The growth situation of the strain is shown in Figure 5 . Under aerobic conditions, in DM culture medium, YB082 could grow using ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen as nitrogen sources respectively. After 120 h, the biomass (OD600) was 7.3 times ( Figure 5 A), 1.9 times ( Figure 5 B), and 2.7 times ( Figure 5 C) of that at the initial stage of culture respectively. It can be seen that when ammonia nitrogen and nitrite nitrogen are used as nitrogen sources in DM culture medium, YB082 has better growth activity, grows rapidly, accumulates a large amount of biomass, and ammonia nitrogen is the most suitable nitrogen source.
[0046] Under anaerobic conditions, in DM culture medium, YB082 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 1.4 times ( Figure 5 E) and 1.3 times ( Figure 5 F) of that at the initial culture. The growth rate is relatively small. The biomass of ammonia nitrogen decreased instead of increasing, being 0.9 times ( Figure 5 D) of that at the initial culture.
[0047] The nitrogen removal ability of strain YB082 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 446 mg / L, 154 mg / L, and 720 mg / L respectively, after 120 hours, the removal rates of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen are 22.3% ( Figure 6 A), 89.4% ( Figure 6 B), and 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 445 mg / L, 153 mg / L, and 719 mg / L, after YB082 is cultured for 120 hours, the removal rates of nitrate nitrogen and nitrite nitrogen are 88.2% ( Figure 6 E) and 100% ( Figure 6 F) respectively. However, ammonia nitrogen cannot be removed ( Figure 6 D).
[0048] In summary, YB082 can tolerate high concentrations of ammonia nitrogen and can remove 22.3% of 446 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 89% 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 YB082 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 anoxic 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 0.4 for YB082, after 67 hours of culture, under anaerobic, aerobic, and anoxic conditions, the cell concentrations reached 0.76, 2.18, and 1.64 respectively, increasing by 1.9 times ( Figure 7 A), 5.5 times (Figure 7 B) and 4.3 times ( Figure 7 C). It can be seen that this strain can grow under anaerobic, aerobic and anoxic conditions when the nutritional conditions are sufficient. The growth is best under aerobic conditions and second best under anoxic conditions.
[0051] Example 5 Growth characteristics of the strain under different initial pH conditions The YB082 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] YB082 can grow in a relatively wide pH range. 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 - 4.2. The biomass of the strain is the highest when the initial pH is 8 and 9, and the cell concentrations (OD600) are 4.0 and 4.2 respectively.
[0053] Example 6 Growth characteristics of the strain under different salt concentration conditions The YB082 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] YB082 can tolerate a NaCl concentration of 7% 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%, 5%, and 7% NaCl is 10.2 times, 4.4 times, 2.9 times, and 1.9 times that of the initial culture biomass respectively. The biomass of the strain treated with 9% NaCl decreases, only being 0.9 times that of the initial strain concentration.
Claims
1. A bacterial strain, Alcaligenes odorifaciens YB082, classified and named Alcaligenes defragrans YB082, is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China, and the deposit number CCTCC M20231957, and the deposit date being October 20, 2023.
2. The strain according to claim 1, wherein: 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 denitrification process of 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 Alcaligenes odorifaciens YB082 described in claim 1 or 2, and the taxonomic name Alcaligenes defragrans is YB082, which is deposited in the China Center for Type Culture Collection, with the deposit address being Wuhan University, Wuhan, China, the deposit number being CCTCC M 20231957, and the deposit date being October 20, 2023.
9. The biological denitrification agent according to claim 8, wherein 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, characterized in that, Use of the said strain in removing one or more of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in nitrogen-containing sewage, and the denitrification process of 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.