Efficient aerobic denitrification strain as well as screening method and application thereof
By screening out the highly efficient aerobic denitrifying strain of denitrified, the denitrification of chromatinaceae was converted into nitrogen under aerobic conditions, which solved the secondary pollution and high energy consumption of traditional wastewater denitrification technology, and achieved efficient and environmentally friendly wastewater treatment.
Patent Information
- Application Number
- CN202510447408.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-10
- Publication Date
- 2025-07-01
AI Technical Summary
Traditional wastewater nitrogen removal technology has problems such as secondary pollution, high energy consumption, complex operation and long start-up cycle.
A highly efficient aerobic denitrification strain Achromobacter denitrificans was screened to convert nitrate into nitrogen under aerobic conditions to avoid the production of ammonia nitrogen or nitroso nitrogen intermediates, and has the ability to efficiently denitrogenate, adapting to an environment of 28-32℃ and pH 6.5-8.0.
It has achieved efficient removal of nitrates in water under aerobic conditions, avoided the production of harmful intermediates, and has low toxicity and stability. It is suitable for agricultural, industrial and urban sewage treatment, and has environmentally friendly and economical wastewater treatment effects.
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Abstract
Description
Technical Field
[0001] The present invention relates to the field of microorganisms, and specifically to a highly efficient aerobic denitrifying strain, a screening method thereof, and an application thereof. Background Art
[0002] With the continuous advancement of industrialization and agricultural intensification, the cross-media migration and accumulation of inorganic nitrogen pollutants in water bodies have become a global ecological and environmental problem. Therefore, the problem of excessive nitrogen content needs to be solved urgently. In the field of wastewater denitrification, traditional technologies face challenges due to technical limitations and environmental risks. The breakpoint chlorination method relies on excessive chemical agents (such as liquid chlorine), which easily generates carcinogenic by-products such as chloramines and trihalomethanes, posing a potential risk of secondary pollution; the ammonia stripping method requires high energy consumption to adjust the pH to alkaline and strip ammonia nitrogen, and the efficiency decreases significantly at low temperatures, and the released ammonia gas may exacerbate air pollution; although shortcut nitrification and denitrification (SHARON) and anaerobic ammonium oxidation (Anammox) have theoretical advantages, the former has strict requirements for precise control of temperature (30-40 °C) and dissolved oxygen, and the latter has a long startup period (several months) and the bacterial community is sensitive to the nitrite concentration, both of which limit their engineering applicability. Summary of the Invention
[0003] Aiming at the deficiencies of the prior art, the present invention provides a highly efficient aerobic denitrifying strain, a screening method thereof, and an application thereof, solving the problems of secondary pollution, high energy consumption, complex operation, and long startup period existing in traditional wastewater denitrification technologies.
[0004] To achieve the above object, the present invention is realized through the following technical solutions: A highly efficient aerobic denitrifying strain, the strain is Achromobacter denitrificans, its preservation number is: CGMCC-NO.33861, the strain name is AD-3, the taxonomic name is Achromobacter denitrificans, and the Latin name is Achromobacter denitrificans. It was preserved in the China General Microbiological Culture Collection Center (CGMCC) on March 18, 2025, at the address: No. 3, Courtyard 1, Beichen Fourth Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with a postal code of 100101. It is used to convert nitrate into nitrogen under aerobic conditions, has the ability of denitrification, and does not produce ammonia nitrogen or nitrite nitrogen intermediate products during the denitrification process. This strain has the ability to remove more than 80% of nitrate after screening and can grow stably under the conditions of temperature 28-32 °C and pH 6.5-8.0.
[0006] The strain is Achromobacter denitrificans, which can convert nitrate in water into nitrogen under aerobic conditions. The core advantage of this process is the ability to effectively remove nitrate nitrogen from water without generating harmful intermediate products such as ammonia nitrogen and nitrite nitrogen during the denitrification process. Under aerobic conditions, Achromobacter denitrificans can efficiently utilize nitrate as an electron acceptor to carry out reduction reactions, directly producing harmless nitrogen gas, avoiding the problem of ammonia nitrogen accumulation that may occur in traditional treatment methods. The excellent performance of this strain stems from its inherent denitrifying enzyme system, which can efficiently reduce nitrate under sufficient oxygen conditions.
[0007] A screening method for a highly efficient aerobic denitrifying strain includes the following steps:
[0008] S1. Isolate Achromobacter denitrificans from sludge:
[0009] Place the selected sludge in a culture flask containing a denitrification medium and conduct shaking culture to enrich aerobic denitrifying strains;
[0010] Under the condition of shaking culture, oxygen is continuously supplied to the culture solution, providing sufficient oxygen for the growth of denitrifying strains. Under this condition, the aerobic metabolism of Achromobacter denitrificans is fully activated, and it can promote its rapid growth and complete the denitrification process through the processes of oxidizing organic matter and reducing nitrate.
[0011] S2. Isolate the strain and conduct single colony culture:
[0012] Dilute the culture solution by different multiples and spread it on an agar plate containing a separation medium, then inoculate the strain and place it in an incubator for culture to obtain single colony strains;
[0013] Through dilution and spread culture, the number of colonies on each plate can be effectively reduced, so that each colony originates from a single cell. On a specific medium, only strains with strong denitrification ability can grow and reproduce, thus ensuring that the selected strains have a high denitrification efficiency.
[0014] S3. Select and culture the obtained strains:
[0015] Inoculate the strain into a liquid denitrification medium for further culture, and observe the removal effect of different strains on NO3 - -N, and select the strain with the highest nitrate removal efficiency for standby;
[0016] In a liquid medium, the strain reduces nitrate to nitrogen gas through its intrinsic denitrification enzyme system. During this process, nitrate serves as an electron acceptor and is reduced to nitrogen gas in an aerobic environment. The efficiency of this reaction is affected by the metabolic activity of the strain. Strains with high nitrate removal efficiency exhibit strong denitrification ability and can rapidly remove nitrogen pollution in water through this process.
[0017] S4. Observe the morphology of the reserve strains through a scanning electron microscope;
[0018] Scanning electron microscope observation can reveal the microscopic morphological characteristics of the strain and help confirm whether the selected strain is the expected Achromobacter denitrificans. These morphological characteristics are closely related to its biological functions and contribute to the study of the strain's adaptability and growth characteristics in the environment.
[0019] S5. Conduct molecular biological identification of the reserve strains.
[0020] Gene sequencing and BLAST alignment can help confirm the species of the strain and provide accurate molecular information for subsequent application research. Through molecular biological methods, phylogenetic analysis of the strain can be carried out to ensure that it is Achromobacter denitrificans and further verify its potential nitrogen removal characteristics.
[0021] Preferably, the shaking culture in S1 is carried out at a temperature of 30 °C, and the shaking speed is set at 140 - 160 rpm, and the culture time is 35 - 37 h.
[0022] Preferably, in S2, the dilution factor is 10 -1 to 10 -7 , and 190 - 210 μL of each dilution is sampled and spread on an agar plate containing a separation medium. The spreading thickness is 0.3 - 0.4 mm, and then it is cultured in an incubator at a temperature of 36 - 38 °C for 71 - 73 h.
[0023] Preferably, in S3, the inoculation density is 1 - 3% v / v, and it is cultured at a temperature of 28 - 32 °C for 24 - 48 h.
[0024] Preferably, in S4, the selected strain is inoculated into a denitrification medium and cultured to the logarithmic phase at 29 - 31 °C and 140 - 160 r / min;
[0025] Subsequently, the strain in the logarithmic phase is taken, washed with sterile water and the supernatant is removed, and fixed with 2 - 3% glutaraldehyde at a temperature of 3 - 5 °C for 11.5 - 12.5 h;
[0026] After fixation, the strain is dehydrated using a freeze dryer, and finally the morphological characteristics of the strain are observed through a scanning electron microscope.
[0027] Preferably, in S5, after extraction and dehydration, the genomic DNA of the strain is extracted using a DNA extraction kit.
[0028] The extracted genomic DNA is subjected to PCR amplification using the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-ACGGCTACCTTGTTACGACT-3′).
[0029] After the amplification is completed, the amplification product is subjected to gene sequencing, followed by BLAST alignment in the NCBI database, and a phylogenetic tree is constructed using MEGA11 software.
[0030] A screening method for a highly efficient aerobic denitrifying strain and the application of the screened denitrifying strain in wastewater treatment.
[0031] The present invention provides a highly efficient aerobic denitrifying strain, its screening method and application. It has the following
[0032] Beneficial effects:
[0033] In the present invention, Achromobacter denitrificans converts nitrate in water into nitrogen under aerobic conditions, avoiding the harmful intermediate products of ammonia nitrogen and nitrite nitrogen that may be produced in traditional treatment methods, and ensuring the environmental protection of the wastewater treatment process. In addition, Achromobacter denitrificans has low toxicity, can grow stably in various wastewater environments, has little impact on the ecosystem, and ensures the safety of the treatment process. At the same time, it can adapt to the treatment requirements of different types of wastewater, such as agricultural wastewater, industrial wastewater and municipal sewage, etc., and has a wide application prospect. The high-efficiency metabolic characteristics of the strain enable it to complete the removal of nitrate in a short time and operate stably within a wide range of temperatures and pH values, which is an environmentally friendly and economical wastewater treatment solution. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 It is a schematic flow chart of the method of the present invention;
[0035] Figure 2 It is a scanning electron micrograph of the strain of the present invention;
[0036] Figure 3 It is a phylogenetic tree diagram of the strain of the present invention;
[0037] Figure 4 It is the test result of the denitrification and nitrogen removal performance of the strain of the present invention under different culture conditions Figure 1 ;
[0038] Figure 5 It is the test result of the denitrification and nitrogen removal performance of the strain of the present invention under different culture conditions Figure 2 . Detailed implementation manners
[0039] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the drawings in the specification of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts belong to the scope of protection of the present invention.
[0040] For a better understanding of the present invention, the above content will be described in detail below in conjunction with specific embodiments.
[0041] Please refer to the attached Figures 1 - 5 :
[0042] Example 1:
[0043] S1. Place the selected sludge in a culture flask containing a denitrification medium, and perform shaking culture at a shaking speed of 150 rpm and a temperature of 30 °C for 36 h to enrich aerobic denitrifying strains;
[0044] S2. Dilute the culture solution successively by multiples of 10 -1 to 10 -7 . Take 200 μL of each diluted sample and coat it on an agar plate containing a separation medium with a thickness of 0.35 mm, and then culture it in an incubator at a temperature of 37 °C for 72 h;
[0045] S3. Select the strains obtained in S2 and inoculate them into a liquid denitrification medium at an inoculation density of 2% v / v, and culture them at a temperature of 30 °C for 36 h, and select the strains with the highest nitrate removal efficiency for standby;
[0046] S4. Inoculate the selected strains into a denitrification medium, and culture them at 30 °C and 150 r / min until the logarithmic phase; then take the strains in the logarithmic phase, wash them with sterile water and remove the supernatant, and fix them with 2.5% glutaraldehyde at a temperature of 4 °C for 12 h; after fixation, dehydrate the strains using a freeze dryer, and finally observe the morphological characteristics of the strains through a scanning electron microscope.
[0047] S5. Extract the genomic DNA of the dehydrated strains using a DNA extraction kit; use the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-ACGGCTACCTTGTTACGACT-3′) to perform PCR amplification on the extracted genomic DNA; after the amplification is completed, sequence the amplification products, and then perform BLAST alignment in the NCBI database, and use MEGA11 software to construct a phylogenetic tree.
[0048] Example 2:
[0049] S1. Place the selected sludge in a culture flask containing denitrification medium, and perform shaking culture for 35 h at a shaking speed of 140 rpm and a temperature of 30 °C to enrich aerobic denitrifying strains;
[0050] S2. Dilute the culture solution successively by multiples of 10 -1 to 10 -7 Take 190 μL of each diluted sample and spread it on an agar plate containing separation medium with a thickness of 0.3 mm, and then culture it in an incubator at a temperature of 36 °C for 71 h;
[0051] S3. Select the strains obtained in S2 and inoculate them into the liquid denitrification medium at an inoculation density of 1% v / v, and culture them at a temperature of 28 °C for 24 h, and select the strains with the highest nitrate removal efficiency for standby;
[0052] S4. Inoculate the selected strains into the denitrification medium, and culture them at 29 °C and 140 r / min until the logarithmic phase; then take the strains in the logarithmic phase, wash them with sterile water and remove the supernatant, fix them with 2% glutaraldehyde at a temperature of 3 °C for 11.5 h; after fixation, dehydrate the strains with a freeze dryer, and finally observe the morphological characteristics of the strains by scanning electron microscopy;
[0053] S5. Extract the genomic DNA of the dehydrated strains using a DNA extraction kit; use the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-ACGGCTACCTTGTTACGACT-3′) to perform PCR amplification on the extracted genomic DNA; after the amplification is completed, sequence the amplified product, then perform BLAST comparison in the NCBI database, and use MEGA11 software to construct a phylogenetic tree.
[0054] Example 3:
[0055] S1. Place the selected sludge in a culture flask containing denitrification medium, and perform shaking culture for 37 h at a shaking speed of 160 rpm and a temperature of 30 °C to enrich aerobic denitrifying strains;
[0056] S2. Dilute the culture solution successively by multiples of 10 -1 to 10 -7 Take 210 μL of each diluted sample and spread it on an agar plate containing separation medium with a thickness of 0.4 mm, and then culture it in an incubator at a temperature of 38 °C for 73 h;
[0057] S3. Select the strains obtained in S2 and inoculate them into the liquid denitrification medium at an inoculation density of 3% v / v, and culture them at a temperature of 32 °C for 48 h. Select the strain with the highest nitrate removal efficiency for standby;
[0058] S4. Inoculate the selected strains into the denitrification medium and culture them to the logarithmic phase at 31 °C and 160 r / min; Subsequently, take the strains in the logarithmic phase, wash them with sterile water and remove the supernatant, and fix them with 3% glutaraldehyde at a temperature of 5 °C for 12.5 h; After fixation, dehydrate the strains using a freeze dryer, and finally observe the morphological characteristics of the strains through a scanning electron microscope;
[0059] S5. Extract the genomic DNA of the strains after dehydration treatment using a DNA extraction kit; Use the universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-ACGGCTACCTTGTTACGACT-3′) to perform PCR amplification on the extracted genomic DNA; After the amplification is completed, sequence the amplification products, then perform BLAST alignment in the NCBI database, and use MEGA11 software to construct a phylogenetic tree.
[0060] Although the embodiments of the present invention have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. A highly efficient aerobic denitrifying strain, characterized in that: include: The strain is a denitrifying colorless bacterium, with a preservation number of CGMCC-NO.33861 and a classification name of AD-3. It was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (CGMCC) on March 18, 2025. It is used to convert nitrate into nitrogen gas under aerobic conditions and has the ability to denitrify. No ammonia nitrogen or nitrite nitrogen intermediates are produced during the denitrification process. The strain has been screened to have a nitrate removal rate of more than 80%, and can grow stably under conditions of 28-32°C and pH 6.5-8.
0.
2. A method for screening a highly efficient aerobic denitrifying strain, The high-efficiency aerobic denitrification strain according to claim 1, characterized in that: The following steps are involved: S1. Isolation of denitrifying achromobacter from sludge: The selected sludge is placed in a culture bottle containing a denitrification culture medium and cultured by shaking to enrich aerobic denitrification strains; S2. Isolation of strains and cultivation of single colonies: The culture solution is diluted at different times and spread on an agar plate containing a separation medium, then the strain is inoculated and placed in an incubator for cultivation to obtain a single colony strain; S3. Select and culture the obtained strains: The strains were inoculated into liquid denitrification medium for further cultivation, and the effects of different strains on NO3 - -N removal effect, select the strain with the highest nitrate removal efficiency for use; S4, observing the morphology of the standby strains by scanning electron microscopy; S5. Conduct molecular biological identification on the alternative strains.
3. The method for screening a highly efficient aerobic denitrifying strain according to claim 2, characterized in that: The shaking culture in S1 is carried out at a temperature of 30° C., with the shaking speed set at 140-160 rpm and the culture time being 35-37 h.
4. The method for screening a highly efficient aerobic denitrifying strain according to claim 2, characterized in that: In S2, the dilution factor is 10 -1 Up to 10 -7 , and 190-210 μL of each dilution factor was sampled and spread on an agar plate containing a separation medium, the coating thickness was 0.3-0.4 mm, and then cultured at a temperature of 36-38° C. for 71-73 h in an incubator parameter.
5. The method for screening a highly efficient aerobic denitrifying strain according to claim 2, characterized in that: In the S3, the inoculation density is 1-3% v / v, and the culture is carried out at a temperature of 28-32° C. for 24-48 hours.
6. The method for screening a highly efficient aerobic denitrifying strain according to claim 2, characterized in that: In S4, the selected strain is inoculated into a denitrification medium and cultured at 29-31° C. and 140-160 r / min until the logarithmic phase; Then, the strain in the logarithmic phase was taken, washed with sterile water, the supernatant was removed, and fixed with 2-3% glutaraldehyde at a temperature of 3-5°C for 11.5-12.5h; After fixation, the strain was dehydrated using a freeze dryer, and finally the morphological characteristics of the strain were observed using a scanning electron microscope.
7. The method for screening a highly efficient aerobic denitrifying strain according to claim 2, characterized in that: In S5, the genomic DNA of the strain is extracted using a DNA extraction kit after the dehydration treatment; The extracted genomic DNA was subjected to PCR amplification using universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-ACGGCTACCTTGTTACGACT-3′); After amplification, the amplified products were sequenced, followed by BLAST comparison of the NCBI database, and a phylogenetic tree was constructed using MEGA11 software.
8. A method for screening a highly efficient aerobic denitrifying strain according to any one of claims 2 to 7, and use of the screened denitrifying strain in wastewater treatment.