Denitrification and phosphorus removal bacterial agent, preparation method and application thereof

Abstract

The present application relates to a kind of denitrification phosphorus removal bacterial agent and its preparation method and application, the bacterial agent includes acinetobacter pittii ( Acinetobacter pittii ) PA6 and pseudomonas aeruginosa ( Pseudomonas aeruginosa ), wherein the preservation number of acinetobacter pittii ( Acinetobacter pittii ) PA6 is CGMCC No.25181.The bacterial agent provided by the present application can efficiently remove total nitrogen and total phosphorus in sewage under the same condition, and will not cause secondary pollution in long-term use.

Description

Technical Field

[0001] This invention belongs to the field of environmental pollution control technology, specifically relating to a denitrifying and phosphorus-removing bacterial agent, its preparation method, and its application. Background Technology

[0002] Traditional biological nitrogen and phosphorus removal processes are achieved through the coexistence of nitrifying bacteria, denitrifying bacteria, and polyphosphate-accumulating bacteria. Traditional biological nitrogen removal involves the oxidation of ammonia nitrogen to nitrite or nitrate by autotrophic nitrifying bacteria and anaerobic denitrifying bacteria, which are then reduced to gaseous nitrogenous products, thus removing nitrogen from wastewater. Traditional biological phosphorus removal utilizes polyphosphate-accumulating bacteria to release phosphorus extracellularly under anaerobic conditions and then absorb it under aerobic conditions. Because the oxygen supply conditions for microorganisms differ in different processes, alternating anaerobic and aerobic conditions is necessary, increasing the operational complexity of the wastewater treatment process. Therefore, a certain degree of contradiction and competition exists, often resulting in the inability to simultaneously achieve optimal nitrogen and phosphorus removal efficiency in practical applications, thus affecting the overall removal effect.

[0003] In recent years, the discovery of heterotrophic nitrifying-aerobic denitrifying polyphosphate-accumulating bacteria has attracted increasing attention. These bacteria can simultaneously remove nitrogen and phosphorus from wastewater under both aerobic and heterotrophic conditions, overcoming the main drawbacks of traditional processes, while also removing a certain amount of carbonaceous organic pollutants. However, in practical applications, the phosphorus load in some wastewater may exceed the phosphorus removal capacity of these denitrifying and phosphorus-removing bacteria, leading to incomplete phosphorus removal.

[0004] CN201610007503.0 discloses a heterotrophic nitrifying-aerobic denitrifying and phosphorus-accumulating Acinetobacter strain and its application in nitrogen and phosphorus removal from wastewater. This strain, Acinetobacter p. WZUF26, is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC NO.9045. The optimal temperature range for nitrogen and phosphorus removal from wastewater by this strain is 15-30℃, exhibiting a relatively wide temperature adaptability. Under suitable conditions, this strain effectively removes NH4+ from artificially treated nitrogen and phosphorus wastewater. + -N, NO3 - -N, NO2 - The removal rates of -N and P were 8.16 mg / (L·h), 6.02 mg / (L·h), 10.76 mg / (L·h), and 0.39 mg / (L·h), respectively, indicating relatively high removal rates. However, when treating livestock and poultry wastewater with a total phosphorus concentration of less than 5 mg / L, the removal rate was less than 60%.

[0005] CN202010549908.3 discloses a composite microbial agent for enhancing nitrogen and phosphorus removal in wastewater and its application. The composite microbial agent comprises the following raw materials in parts by weight: 40-60 parts of Acinetobacter, 20-30 parts of Bacillus, 5-15 parts of biofilm, and 5-25 parts of iron filings. Compared to current nitrogen and phosphorus removal technologies, this microbial agent can achieve highly efficient nitrogen and phosphorus removal from wastewater through the synergistic enhancement of biofilm and iron filings. However, this method enhances the nitrogen and phosphorus removal effect through multiple physical, chemical, and biological processes, and the long-term introduction of iron filings can lead to the accumulation of metal ions in the water. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a nitrogen and phosphorus removal bacterial agent, its preparation method, and its application. The bacterial agent provided by this invention can efficiently remove total nitrogen and total phosphorus from wastewater under the same conditions, and will not cause secondary pollution with long-term use.

[0007] The first aspect of the present invention provides a denitrifying and phosphorus-removing bacterial agent, which includes Acinetobacter pittii PA6 and Pseudomonas aeruginosa, wherein Acinetobacter pittii PA6 has the accession number CGMCC No. 25181.

[0008] In this invention, the Pseudomonas aeruginosa strain used is the type strain, with accession number CGMCC 1.1785, purchased from the China General Microbiological Culture Collection Center.

[0009] In this invention, *Acinetobacter pittii* PA6 was deposited on June 27, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25181, located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Its main morphological characteristics are as follows: after culturing on TSA medium at 37°C for 18 hours, the colonies are yellow, round, moist, opaque, and have neat edges. Under a microscope, the bacteria are rod-shaped, 0.3-0.5 μm × 0.4-1.0 μm, arranged singly or in pairs, and are Gram-negative.

[0010] In the bacterial agent of the present invention, preferably, Acinetobacter pittii PA6 and Pseudomonas aeruginosa are mixed in a bacterial mass ratio of 1-10:1, more preferably 1-3:1.

[0011] The second aspect of this invention provides a method for preparing a nitrogen- and phosphorus-removing bacterial agent, comprising the following steps: inoculating Acinetobacter pittii PA6 and Pseudomonas aeruginosa onto LB solid medium for activation culture; after activation, using an inoculation loop to collect colonies from the plate and inoculating them into LB liquid medium, and aerobically culturing until OD... 660 The OD value is 0.8 or higher, preferably 1.0-1.2, to obtain a liquid bacterial suspension; after collecting the bacterial cells, they are resuspended to adjust the OD value of the bacterial suspension. 660 The concentration is 1.5-2.0, and then mixed according to the required ratio to form a denitrifying and phosphorus-removing bacterial agent.

[0012] In the preparation method of the inoculant of this invention, the LB liquid culture medium formula is: NaCl 5-15 g / L, peptone 5-15 g / L, and yeast extract 3-8 g / L. The solid culture medium is prepared by adding 1%-2% agar to the liquid culture medium.

[0013] In the preparation method of the bacterial agent of the present invention, the activation culture conditions are: temperature of 25-30℃ and culture time of 12-48h.

[0014] In the preparation method of the bacterial agent of the present invention, the aerobic culture conditions are: pH value of 6-9, temperature of 25-30℃, and dissolved oxygen concentration of 0.5-2.0mg / L.

[0015] In the preparation method of the bacterial agent of the present invention, the bacterial cells can be collected by means of filtration, centrifugation, etc.

[0016] In the preparation method of the bacterial agent of the present invention, resuspension can be carried out using culture medium, physiological saline, etc., with physiological saline being preferred.

[0017] In the preparation method of the bacterial agent of the present invention, the bacterial suspensions after resuspending the two bacteria are mixed in a mass ratio of 1-10:1, preferably 1-3:1.

[0018] The third aspect of this invention provides the application of the above-mentioned denitrifying and phosphorus-removing bacterial agent for the simultaneous denitrification and phosphorus removal of wastewater.

[0019] In the application of this invention, it is used to treat wastewater or sewage containing ammonia nitrogen, nitrate nitrogen / nitrite nitrogen, phosphate, and COD. The total nitrogen concentration (the sum of ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen) in the treated wastewater is not higher than 300 mg / L, preferably 50-200 mg / L, the total phosphorus concentration is not higher than 30 mg / L, preferably 10-30 mg / L, and the COD concentration is not higher than 4000 mg / L.

[0020] In the application of this invention, the dosage of the microbial agent is 0.1%-1% by mass fraction, preferably 0.2%-0.6%.

[0021] In the application of this invention, it is best to add a certain amount of carbon source during nitrogen and phosphorus removal. The carbon source can be at least one of sodium acetate, sodium lactate, sodium citrate, sodium succinate, etc., and should be added according to a COD / TN ratio of 10:1-30:1, preferably 15:1-20:1.

[0022] In the application of this invention, the wastewater treatment conditions are generally: temperature of 15-35℃, pH of 6-9, and dissolved oxygen concentration of 0.5-2.0 mg / L.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) The bacterial agent of this invention, through the synergistic effect of Acinetobacter pittii PA6 and Pseudomonas aeruginosa, can achieve efficient removal of total nitrogen and total phosphorus simultaneously under aerobic and heterotrophic conditions. The removal effect is significantly better than that of using a single bacterium alone.

[0025] (2) The formulation of the microbial agent of the present invention is simple, the preparation process is simple, and there is no secondary pollution.

[0026] (3) The bacterial agent of the present invention can be directly used as an inoculum to treat nitrogen and phosphorus wastewater, which solves the competition and adverse effects of nitrifying bacteria, denitrifying bacteria and polyphosphate-accumulating bacteria in the process of nitrogen and phosphorus removal. Detailed Implementation

[0027] The technical solution and application effects of the present invention will be described in detail below with reference to specific embodiments. The embodiments are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures; however, the scope of protection of the present invention is not limited to the following embodiments.

[0028] Unless otherwise specified, the experimental methods used in the following examples are conventional methods in the art. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores.

[0029] In this invention, ammonia nitrogen concentration was determined using GB7478-87 "Determination of Ammonium in Water - Distillation and Titration Method"; nitrate nitrogen concentration was determined using GB7480-1987 "Water Quality - Determination of Nitrate Nitrogen - Phenol Disulfonic Acid Spectrophotometric Method"; nitrite nitrogen concentration was determined using GB7493-87 "Water Quality - Determination of Nitrite Nitrogen - Spectrophotometric Method"; total nitrogen concentration was determined using GB11894-89 "Water Quality - Determination of Total Nitrogen - Alkaline Potassium Persulfate Digestion Ultraviolet Spectrophotometric Method"; total phosphorus concentration was determined using GB11893-89 "Ammonium Molybdate Spectrophotometric Method"; and COD was determined using GB11914-89 "Water Quality - Determination of Chemical Oxygen Demand - Dichromate Method".

[0030] The liquid LB medium used in the bacterial agent of this invention has the following formulation: NaCl 10 g / L, peptone 10 g / L, and yeast extract 5 g / L. The solid medium is the liquid medium with 20 g / L agar added.

[0031] Example 1: Preparation of bacterial agent

[0032] (1) Acinetobacter pittii PA6 and Pseudomonas aeruginosa type strain (CGMCC 1.1785) were inoculated onto LB solid medium, spread evenly, and then placed in a constant temperature incubator at 30℃ for 24h for activation culture.

[0033] (2) Using an inoculation loop, take colonies of the two strains from the plate and inoculate them separately into LB liquid culture medium. Incubate at 30℃, pH 7.0-7.5, and dissolved oxygen 1.0-2.5 mg / L until OD. 660 Set the value to 1.0 to obtain liquid bacterial solution.

[0034] (3) After centrifuging the liquid bacterial suspension, collect the bacterial cells and resuspend them in physiological saline to adjust the OD of the bacterial suspension. 660 The concentration is 1.5, and then mixed according to the required ratio to obtain the denitrifying and phosphorus-removing bacterial agent. The bacterial cell numbering and specific composition are shown in Table 1.

[0035] Table 1 Composition and ratio of the microbial agent

[0036] serial number Microbial agent composition mass ratio 1# Acinetobacter piezoides PA6: Pseudomonas aeruginosa type strain 1:1 2# Acinetobacter piezoides PA6: Pseudomonas aeruginosa type strain 3:1 3# Acinetobacter piezoides PA6: Pseudomonas aeruginosa type strain 5:1 4# Acinetobacter piezoides PA6: Pseudomonas aeruginosa type strain 8:1 5# Acinetobacter piezoides PA6: Pseudomonas aeruginosa type strain 10:1

[0037] Examples 2-6

[0038] A chemical plant generates ammonia- and phosphorus-containing wastewater with an ammonia nitrogen concentration of 150 mg / L, total phosphorus of 29 mg / L, and COD of 3500 mg / L. The original system had poor phosphorus removal efficiency, and long-term operation resulted in an increase in total nitrogen. The bacterial agent prepared in Example 1 (numbered sequentially from Examples 2-6) was added to the system at 0.2% of the wastewater treatment volume, along with sodium acetate at a COD / TN ratio of 15:1. The treatment conditions were: temperature 25℃, pH 7.5-8.5, and dissolved oxygen concentration 0.5-2.0 mg / L. The treatment results after 5 days of treatment are shown in Table 2.

[0039] Table 2 Treatment effects of different microbial agents (unit: mg / L)

[0040]

[0041] As shown in Table 2, the total phosphorus and total nitrogen in the effluent decreased significantly after treatment with the bacterial agent of this invention. However, under the same conditions, treatment with a bacterial agent containing only a single bacterium yielded poor results.

[0042] Examples 7-11

[0043] The laboratory-prepared wastewater contained nitrate nitrogen, nitrite nitrogen, and phosphorus, with nitrate nitrogen concentration of 135 mg / L, nitrite nitrogen concentration of 55 mg / L, total phosphorus of 22 mg / L, and COD of 2508 mg / L. The bacterial agent prepared in Example 1 (numbered sequentially as Examples 7-11) was inoculated into the system at 0.5% of the wastewater treatment volume, and sodium acetate was added simultaneously at a COD / TN ratio of 15:1. The treatment conditions were: temperature 25℃, pH 7-8, and dissolved oxygen concentration 0.5-2.0 mg / L. After 5 days of operation, the treatment results are shown in Table 3.

[0044] Table 3 Treatment effects of different microbial agents (unit: mg / L)

[0045]

[0046] As shown in Table 3, the total phosphorus and total nitrogen in the effluent decreased significantly after treatment with the bacterial agent of this invention. However, under the same conditions, treatment with a bacterial agent containing only a single bacterium yielded poor results.

Claims

1. A denitrifying and phosphorus-removing bacterial agent, characterized in that: This bacterial agent includes Acinetobacter pitera (… Acinetobacter pittii PA6 and Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), including Acinetobacter pitera ( Acinetobacter pittii PA6 has the accession number CGMCC No. 25181; Pseudomonas aeruginosa ( Pseudomonas aeruginosa The type strain used was CGMCC 1.1785; the bacterial agent contained Acinetobacter pietrogenia (…). Acinetobacter pittii PA6 and Pseudomonas aeruginosa ( Pseudomonas aeruginosa Mix at a bacterial mass ratio of 1-10:

1.

2. The microbial agent according to claim 1, characterized in that: Acinetobacter piezophyllum ( Acinetobacter pittii PA6 was deposited on June 27, 2022, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 25181. The deposit address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The main morphological characteristics are as follows: after culturing on TSA medium at 37°C for 18 hours, the colonies are yellow, round, moist, opaque, and have neat edges. Under a microscope, the bacteria are rod-shaped, 0.3-0.5 μm × 0.4-1.0 μm, arranged singly or in pairs, and are Gram-negative.

3. The microbial agent according to claim 1, characterized in that: Pseudomonas aeruginosa ( Pseudomonas aeruginosa Purchased from the China General Microbiological Culture Collection Center.

4. The microbial agent according to claim 1, characterized in that: Acinetobacter piezophyllum (in bacterial agent) Acinetobacter pittii PA6 and Pseudomonas aeruginosa ( Pseudomonas aeruginosa Mix at a bacterial mass ratio of 1-3:

1.

5. A method for preparing the denitrifying and phosphorus-removing bacterial agent according to any one of claims 1-4, characterized in that... Includes the following: Acinetobacter pitera ( Acinetobacter pittii PA6 and Pseudomonas aeruginosa ( Pseudomonas aeruginosa The colonies were inoculated separately onto LB solid medium for activation culture; after activation, colonies were taken from the plates using an inoculation loop and inoculated separately onto LB liquid medium, and cultured aerobically until OD. 660 A liquid bacterial suspension was obtained with an OD value of 0.8 or higher; the bacterial cells were collected and resuspended to adjust the OD value of the suspension. 660 The concentration is 1.5-2.0, and then mixed according to the required ratio to form a denitrifying and phosphorus-removing bacterial agent.

6. The preparation method according to claim 5, characterized in that: Aerobic culture to OD 660 The concentration was set to 1.0-1.2 to obtain liquid bacterial solution.

7. The preparation method according to claim 5, characterized in that: The LB liquid culture medium formula is as follows: NaCl 5-15 g / L, peptone 5-15 g / L, yeast extract 3-8 g / L.

8. The preparation method according to claim 5, characterized in that: The activation culture conditions are: temperature 25-30℃, culture time 12-48h.

9. The preparation method according to claim 5, characterized in that: The aerobic culture conditions are: pH 6-9, temperature 25-30℃, and dissolved oxygen concentration 0.5-2.0 mg / L.

10. The preparation method according to claim 5, characterized in that: Resuspension is performed using at least one of culture medium and physiological saline.

11. The preparation method according to claim 10, characterized in that: Resuspension was performed using physiological saline.

12. The preparation method according to claim 5 or 10, characterized in that: The bacterial suspensions of the two bacteria were mixed at a mass ratio of 1-10:

1.

13. The preparation method according to claim 12, characterized in that: The bacterial suspensions of the two bacteria were mixed at a mass ratio of 1-3:

1.

14. The application of a microbial agent according to any one of claims 1-4 or a microbial agent prepared by the method according to any one of claims 5-13, characterized in that: It is used for simultaneous nitrogen and phosphorus removal from wastewater.

15. The application according to claim 14, characterized in that: This treatment is used for wastewater or sewage containing ammonia nitrogen, nitrate nitrogen / nitrite nitrogen, phosphate, and COD. The total nitrogen concentration in the treated wastewater shall not exceed 300 mg / L, including ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen; the total phosphorus concentration shall not exceed 30 mg / L; and the COD concentration shall not exceed 4000 mg / L.

16. The application according to claim 14, characterized in that: The total nitrogen concentration in the treated wastewater is 50-200 mg / L, and the total phosphorus concentration is 10-30 mg / L.

17. The application according to claim 14, characterized in that: The dosage of microbial agent is 0.1%-1% by mass.

18. The application according to claim 17, characterized in that: The dosage of microbial agent is 0.2%-0.6% by mass fraction.

19. The application according to claim 14, characterized in that: During nitrogen and phosphorus removal, a certain amount of carbon source is added. The carbon source is at least one of sodium acetate, sodium lactate, sodium citrate, and sodium succinate, and is added according to a COD / TN ratio of 10:1-30:

1.

20. The application according to claim 19, characterized in that: The carbon source is added at a COD / TN ratio of 15:1-20:

1.

21. The application according to claim 14, characterized in that: The wastewater treatment conditions are: temperature 15-35℃, pH 6-9, and dissolved oxygen concentration 0.5-2.0 mg / L.

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