Application of heterotrophic nitrification-aerobic denitrification acinetobacter in nitrogen-containing wastewater treatment

Abstract

The invention relates to the technical field of wastewater treatment, in particular to application of heterotrophic nitrification-aerobic denitrification acinetobacter in nitrogen-containing wastewater treatment, and the heterotrophic nitrification-aerobic denitrification acinetobacter AD-5 provided by the invention is a microbial strain with remarkable characteristics and application value. The strain has the most remarkable characteristic that due to the unique nitrogen conversion mechanism, NO2--N and NO3--N can be efficiently used as electron acceptors for respiration under the aerobic condition, and the dependence of a traditional denitrification process on an anaerobic environment is broken through; compared with a conventional biological denitrification strain, the AD-5 strain has the excellent performance advantages that the growth speed is high, the process energy consumption is low, and the operation conditions are flexible; the biggest innovation of the acinetobacter AD-5 provided by the invention is that the synchronous nitrification and denitrification nitrogen removal can be realized, and the special metabolic pathway not only greatly simplifies the traditional multi-section biological nitrogen removal process flow, but also remarkably reduces the treatment cost and the energy consumption.

Description

Technical Field

[0001] The invention relates to the technical field of wastewater treatment, and in particular to application of heterotrophic nitrification-aerobic denitrification Acinetobacter in the treatment of nitrogen-containing wastewater. Background Art

[0002] In recent years, with the acceleration of industrialization and urbanization, the impact of human activities on the water environment has become increasingly serious. The discharge of high-concentration ammonia nitrogen wastewater has become one of the important sources of water pollution. When ammonia nitrogen wastewater enters the water body without proper treatment, it will cause a series of ecological and environmental problems. First of all, excessive concentration of ammonia nitrogen will cause the dissolved oxygen in the water body to continue to decrease, destroying the balance of the water ecosystem. In this case, microorganisms, algae and other aquatic organisms in the water body will face serious threats to their survival, and the ecosystem will suffer great damage. At the same time, ammonia nitrogen pollution will aggravate the eutrophication process of the water body, causing a sharp deterioration of water quality, and thus affecting the overall health of the water ecosystem.

[0003] High ammonia nitrogen wastewater generated during industrial production usually contains complex pollutants, which can be mainly divided into two categories: biodegradable organic matter and difficult-to-degrade pollutants. With the improvement of environmental protection awareness and the continuous improvement of relevant laws and regulations, my country's restrictions on ammonia nitrogen wastewater discharge indicators are becoming more and more stringent. How to achieve efficient and low-cost removal of ammonia nitrogen pollution in water has become a key research direction in the field of water treatment. Traditional treatment methods mainly include physical and chemical treatment technologies, such as stripping, gas lift, breakpoint chlorination, ion exchange, coagulation sedimentation and reverse osmosis. However, these methods generally have the problems of high technical and economic costs and complex treatment processes, and are prone to introduce additional substances such as chloride ions and phosphorus during the treatment process, causing secondary pollution, which greatly limits their practical application value. Summary of the invention

[0004] In view of the deficiencies in the prior art, the object of the present invention is to provide an application of heterotrophic nitrification-aerobic denitrification Acinetobacter in the treatment of nitrogen-containing wastewater.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides an application of a heterotrophic nitrification-aerobic denitrifying Acinetobacter in the treatment of nitrogen-containing wastewater. The heterotrophic nitrification-aerobic denitrifying Acinetobacter is named as Acinetobacter AD-5, which is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC M 20211654.

[0007] In the preferred technical solution of the present invention, the screening and purification steps of Acinetobacter AD-5 are as follows:

[0008] S1. Enrichment: Mix the activated sludge with sterile water, take 1 mL of the sludge suspension and place it in the enrichment medium, and culture it in a constant temperature oscillator at 30°C for 3-5 days;

[0009] S2. Acclimation: Take 1 mL of the above enriched bacterial solution and transfer it to liquid acclimation medium for further culturing. After 24 hours, take the bacterial suspension and dilute it to 10 4 , 10 5 , 10 6 , 10 7 times, take 0.1 mL of each and spread it on the solid acclimation medium, invert and culture it in a 30℃ constant temperature incubator for 3-5 days, until a single colony with clear morphology grows on the surface of the plate, pick single colonies of different morphologies, inoculate them into liquid acclimation medium respectively, and culture them in a constant temperature oscillator at 30℃ and 100-150r / min overnight, and dilute the bacterial suspension grown to the logarithmic growth phase for smearing and culture again, and repeat the above experiment 3-5 times;

[0010] S3, screening: pick single colonies of different morphologies from the diluted and coated acclimation medium, inoculate them into liquid acclimation medium respectively, culture them at 30°C, 100-150r / min constant temperature shaking for 24h, dilute the bacterial solution in gradients and then spread them on BTB medium, culture them in a 30°C constant temperature incubator for 3-5d, pick single colonies that change from yellow to blue in the medium, continue to streak purification on BTB medium, and repeat the operation 3-5 times;

[0011] S4. After multiple enrichment, separation and purification, strains with heterotrophic nitrification or aerobic denitrification properties were screened out, and the screened strains were inoculated into heterotrophic nitrification identification medium and aerobic denitrification identification medium at an inoculation rate of 2%, respectively. After culturing at 30°C and 100-150r / min in a shaking incubator for 24 hours, a high-efficiency heterotrophic nitrification-aerobic denitrification strain was screened out according to the results of water quality detection indicators and named Acinetobacter AD-5.

[0012] In the preferred technical solution of the present invention, in step S1, the formula of the enrichment medium is: 5 g of peptone, 5 g of yeast extract, 10 g of NaCl, and 1 L of distilled water.

[0013] In the preferred technical scheme of the present invention, in step S2, the formula of the acclimation culture medium is: CH3COONa2.93g, KNO30.722g, KH2PO40.088g, MgSO4·7H2O0.2g, trace elements2mL, and distilled water1L; the formula of trace elements is: EDTA15g, ZnSO40.2g, MnCl2·4H2O1.5g, FeSO4·7H2O0.5g, CuSO4·5H2O0.5g, CoCl2·6H2O0.3g, Na2MoO4·2H2O0.2g, CaCl20.1g, and distilled water1L.

[0014] In the preferred technical scheme of the present invention, in step S3, the BTB culture medium formula is: CH3COONa2.93g, KNO30.722g, KH2PO40.088g, MgSO4·7H2O0.2g, trace elements2mL, 2% agar, 1% BTB, distilled water1L; the trace element formula is: EDTA15g, ZnSO40.2g, MnCl2·4H2O1.5g, FeSO4·7H2O0.5g, CuSO4·5H2O0.5g, CoCl2·6H2O0.3g, Na2MoO4·2H2O0.2g, CaCl20.1g, distilled water1L.

[0015] In the preferred technical scheme of the present invention, in step S4, the formula of aerobic nitrification identification culture medium is: CH3COONa 0.937g, KNO3 0.288g, KH2PO4 0.0351g, MgSO4·7H2O 0.2g, trace elements 2mL, and distilled water 1L; the trace element formula is: EDTA 15g, ZnSO4 0.2g, MnCl2·4H2O1.5g, FeSO4·7H2O 0.5g, CuSO4·5H2O 0.5g, CoCl2·6H2O 0.3g, Na2MoO4·2H2O0.2g, CaCl2 0.1g, and distilled water 1L.

[0016] In the preferred technical scheme of the present invention, in step S4, the formula of heterotrophic nitrification identification medium is: CH3COONa 0.937g, NH4Cl 0.153g, KH2PO4 0.0351g, MgSO4·7H2O 0.2g, trace elements 2mL, and distilled water 1L; the trace element formula is: EDTA 15g, ZnSO4 0.2g, MnCl2·4H2O1.5g, FeSO4·7H2O 0.5g, CuSO4·5H2O 0.5g, CoCl2·6H2O 0.3g, Na2MoO4·2H2O0.2g, CaCl2 0.1g, and distilled water 1L.

[0017] The present invention also provides a bacterial agent, comprising fermenting the above-mentioned Acinetobacter AD-5 to obtain a fermentation liquid.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The heterotrophic nitrification-aerobic denitrification Acinetobacter AD-5 provided by the present invention is a microbial strain with significant characteristics and application value. The most significant feature of the strain is its unique nitrogen conversion mechanism, which can efficiently use NO2--N and NO3--N as electron acceptors for respiration under aerobic conditions, breaking through the dependence of traditional denitrification processes on anaerobic environments; compared with conventional biological denitrification strains, the AD-5 strain exhibits outstanding performance advantages: fast growth rate, low process energy consumption, and flexible operating conditions; the greatest innovation of the Acinetobacter AD-5 provided by the present invention is that it can achieve simultaneous nitrification and denitrification, and this special metabolic pathway not only greatly simplifies the traditional multi-stage biological denitrification process, but also significantly reduces the treatment cost and energy consumption; this strain provides a more economical and efficient biotechnology solution for high-ammonia nitrogen sewage treatment, has important theoretical research value and practical engineering application prospects, and is expected to form a technological breakthrough in the field of sewage treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a scanning electron micrograph of the Acinetobacter AD-5 strain provided in Example 1 of the present invention.

[0021] Figure 2 This is a diagram of the heterotrophic nitrification performance of Acinetobacter AD-5 provided in Example 2 of the present invention.

[0022] Figure 3 This is a graph showing the aerobic denitrification performance of Acinetobacter AD-5 provided in Example 3 of the present invention. DETAILED DESCRIPTION

[0023] The present invention is further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.

[0024] It should be noted that, unless otherwise specified, the chemical reagents involved in the present invention were purchased through commercial channels.

[0025] Example 1

[0026] The steps for screening and purification of heterotrophic nitrification-aerobic denitrification Acinetobacter AD-5 are as follows:

[0027] S1. Enrichment: Mix 10 mL of activated sludge with 90 mL of sterile water, take 1 mL of the sludge suspension and place it in the enrichment medium, and culture it in a constant temperature oscillator at 30°C and 120 r / min for 3 days;

[0028] The formula of the enrichment medium is: 5 g peptone, 5 g yeast extract, 10 g NaCl, and 1 L distilled water.

[0029] S2. Acclimation: Take 1 mL of the above enriched bacterial solution and transfer it to liquid acclimation medium for further culturing. After 24 hours, take the bacterial suspension and dilute it to 10 4 , 10 5 , 10 6 , 10 7 times, take 0.1 mL of each and spread it on the solid acclimation medium, and invert and culture it in a 30℃ constant temperature incubator for 3 days until a single colony with clear morphology grows on the surface of the plate; pick single colonies of different morphologies, inoculate them into liquid acclimation medium respectively, and culture them in a constant temperature oscillator at 30℃ and 120r / min overnight, and dilute the bacterial suspension grown to the logarithmic growth phase (OD600=1.0) for smearing culture again, and repeat the above experimental operation 3 times;

[0030] The formula of acclimation culture medium is: CH3COONa 2.93g, KNO3 0.722g, KH2PO4 0.088g, MgSO4·7H2O0.2g, trace elements 2mL, and distilled water 1L; the formula of trace elements is: EDTA 15g, ZnSO4 0.2g, MnCl2·4H2O 1.5g, FeSO4·7H2O 0.5g, CuSO4·5H2O 0.5g, CoCl2·6H2O 0.3g, Na2MoO4·2H2O 0.2g, CaCl20.1g, and distilled water 1L.

[0031] S3, screening: pick single colonies of different morphologies from the acclimation medium that was diluted and coated three times, inoculate them into liquid acclimation medium respectively, and culture them at 30°C and 120r / min constant temperature shaking for 24h; dilute the bacterial solution in gradients (10 -4 -10 -7 ) and then spread on BTB medium, culture in a constant temperature incubator at 30°C for 3 days, pick out the single colony that changes from yellow to blue in the medium, continue to purify by streaking on BTB medium, and repeat this process 4 times;

[0032] The formula of BTB culture medium is: CH3COONa 2.93g, KNO3 0.722g, KH2PO4 0.088g, MgSO4·7H2O0.2g, trace elements 2mL, 2% agar, 1% BTB, and distilled water 1L; the formula of trace elements is: EDTA 15g, ZnSO4 0.2g, MnCl2·4H2O 1.5g, FeSO4·7H2O 0.5g, CuSO4·5H2O 0.5g, CoCl2·6H2O 0.3g, Na2MoO4·2H2O0.2g, CaCl2 0.1g, and distilled water 1L.

[0033] S4. After multiple enrichment, separation and purification, strains with heterotrophic nitrification or aerobic denitrification performance were screened out, and the screened strains were inoculated into heterotrophic nitrification identification medium and aerobic denitrification identification medium at an inoculum of 2%, respectively. After culturing at 30°C and 120 r / min in a shaking incubator for 24 hours, a highly efficient aerobic denitrification strain was screened out according to the results of water quality test indicators and named Acinetobacter AD-5;

[0034] The formula of aerobic nitrification identification medium is: CH3COONa 0.937g, KNO3 0.288g, KH2PO40.0351g, MgSO4·7H2O 0.2g, trace elements 2mL, distilled water 1L; the formula of trace elements is: EDTA 15g, ZnSO4 0.2g, MnCl2·4H2O 1.5g, FeSO4·7H2O 0.5g, CuSO4·5H2O 0.5g, CoCl2·6H2O 0.3g, Na2MoO4·2H2O0.2g, CaCl2 0.1g, distilled water 1L;

[0035] The formula of heterotrophic nitrification identification culture medium is: CH3COONa 0.937g, NH4Cl 0.153g, KH2PO40.0351g, MgSO4·7H2O 0.2g, trace elements 2mL, and distilled water 1L; the trace element formula is: EDTA 15g, ZnSO4 0.2g, MnCl2·4H2O 1.5g, FeSO4·7H2O 0.5g, CuSO4·5H2O 0.5g, CoCl2·6H2O 0.3g, Na2MoO4·2H2O0.2g, CaCl2 0.1g, and distilled water 1L.

[0036] The target strain isolated and purified in Example 1 was inoculated into an enrichment medium for activation and cultured to the logarithmic growth phase. Fresh cells were collected and pre-treated with PBS rinsing, glutaraldehyde fixation, ethanol gradient dehydration, isoamyl acetate substitution, and freeze drying. The morphology of the strain was then observed using a scanning electron microscope (SEM). The results are as follows: Figure 1 As shown, from Figure 1 It can be seen that the strain screened by the present invention is Clostridium.

[0037] The Acinetobacter sp. AD-5 screened by the present invention was deposited in the China Center for Type Culture Collection (CCTCC for short, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province) on December 21, 2021, with the collection number: CCTCC M 20211654, and has been disclosed in Chinese patent document CN202210361200.4.

[0038] Example 2

[0039] Identification of heterotrophic nitrification performance

[0040] The strain was inoculated into 100 mL of sterilized LB medium with an inoculation loop, placed in a constant temperature shaking bed, cultured at 120 r / min, 30°C for 24 h, centrifuged to collect the bacteria, and diluted with sterile water to OD 600 The value is 1.0, and the mixed solution is used as the bacterial solution for subsequent experiments.

[0041] The above experimental strain culture solution was inoculated with 2% inoculum (v / v) into 100 mL of heterotrophic nitrification identification medium, and cultured at 30°C and 120 r / min shaking for 24 h. The OD600 and NH4 + -N、NO3 - -N, NO2 - -Concentration changes of water quality indicators such as N and TN.

[0042] NH4 + -N (initial concentration 40 mg / L) as the sole nitrogen source and CH3COONa as the carbon source (C / N=8) were used to evaluate the heterotrophic nitrification performance of strain AD-5. Figure 2 As shown, Figure 2 The results showed that the growth and metabolism of the strain in the nitrification medium under aerobic conditions. The results showed that within the first three hours, the OD 600 The values ​​are all less than 0.15, NH4 + -N concentration did not show a significant decrease; after a three-hour adaptation period, the strain grew rapidly and entered the logarithmic growth phase at 3h. + -N concentration decreased rapidly, with the maximum NH4 + -N removal rate was 3.66 mg / h; after 18 h, strain OD 600 Reaching a relatively stable value, NH4 + The -N concentration continued to drop to a minimum value, reaching 1.75 mg / L.

[0043] Depend on Figure 2 It can be seen that strain AD-5 did not produce NO2 during the entire denitrification process. - -N and NO3 - -N accumulation, which indicates that NO2 - -N and NO3 - -N may be rapidly converted as an intermediate product, and the TN removal rate within 24 hours was 95.21%, which shows that AD-5 has the ability of synchronous denitrification. While the heterotrophic nitrification reaction is taking place, aerobic denitrification also occurs. AD-5 has good heterotrophic nitrification performance.

[0044] Example 3

[0045] Aerobic denitrification performance evaluation

[0046] The strain was inoculated into 100 mL of sterilized LB medium with an inoculation loop, placed in a constant temperature shaking bed, cultured at 120 r / min, 30°C for 24 h, centrifuged to collect the bacteria, and diluted with sterile water to OD 600 The value is 1.0, and the mixed solution is used as the bacterial solution for subsequent experiments.

[0047] The above experimental strain culture solution was inoculated with 2% inoculum (v / v) in 100 mL of aerobic denitrification identification medium, and cultured at 30 ° C and 120 r / min shaking for 24 h. The OD600 and NH4 + -N、NO3 - -N, NO2 - -Concentration changes of water quality indicators such as N and TN.

[0048] NO3 - -N (initial concentration 40 mg / L) as the sole nitrogen source and CH3COONa as the carbon source (C / N=8) were used to evaluate the aerobic denitrification performance of strain AD-5. Figure 3 As shown, the adaptation period of the strain is the same as that of heterotrophic nitrification, but with NH4 + -N as nitrogen source, compared with the OD of strains at different stages 600 During the logarithmic growth period, as the strain grows rapidly, NO3 - -N concentration decreased rapidly, with the maximum NO3 - -N removal rate was 3.24 mg / h. After 15 h, the strain growth gradually entered a stable period. NO3 - -N concentration continued to drop to the minimum value, reaching 10.67mg / L. During the growth and reproduction of the strain, there was a trace amount of NH4 + -N was detected, which may be due to the release of some NH4 +-N; The TN removal rate of AD-5 reached 71.7% within 24 hours, which indicated that the strain had the ability of aerobic denitrification.

[0049] Finally, it should be noted that the above embodiments do not limit the present invention in any form. For those skilled in the art, some modifications and improvements can be made to the present invention. Therefore, any modification or improvement made without departing from the spirit of the present invention belongs to the scope of protection claimed in the present invention.

Claims

1. An application of heterotrophic nitrification-aerobic denitrification Acinetobacter in the treatment of nitrogen-containing wastewater, characterized in that: The heterotrophic nitrification-aerobic denitrification Acinetobacter is named Acinetobacter AD-5 and is deposited in the China Center for Type Culture Collection with a deposit number of CCTCC M 20211654.

2. The use according to claim 1, characterized in that: The screening and purification steps of the Acinetobacter AD-5 are as follows: S1. Enrichment: Mix the activated sludge with sterile water, take 1 mL of the sludge suspension and place it in the enrichment medium, and culture it in a constant temperature oscillator at 30°C for 3-5 days; S2. Acclimation: Take 1 mL of the above enriched bacterial solution and transfer it to liquid acclimation medium for further culturing. After 24 hours, take the bacterial suspension and dilute it to 10 4 , 10 5 , 10 6 , 10 7 times, take 0.1 mL of each and spread it on the solid acclimation medium, invert and culture it in a 30℃ constant temperature incubator for 3-5 days, until a single colony with clear morphology grows on the surface of the plate, pick single colonies of different morphologies, inoculate them into liquid acclimation medium respectively, and culture them in a constant temperature oscillator at 30℃ and 100-150r / min overnight, and dilute the bacterial suspension grown to the logarithmic growth phase for smearing and culture again, and repeat the above experiment 3-5 times; S3, screening: pick single colonies of different morphologies from the diluted and coated acclimation medium, inoculate them into liquid acclimation medium respectively, culture them at 30°C, 100-150r / min constant temperature shaking for 24h, dilute the bacterial solution in gradients and then spread them on BTB medium, culture them in a 30°C constant temperature incubator for 3-5d, pick single colonies that change from yellow to blue in the medium, continue to streak purification on BTB medium, and repeat the operation 3-5 times; S4. After multiple enrichment, separation and purification, strains with heterotrophic nitrification or aerobic denitrification properties were screened out, and the screened strains were inoculated into heterotrophic nitrification identification medium and aerobic denitrification identification medium at an inoculation rate of 2%, respectively. After culturing at 30°C and 100-150r / min in a shaking incubator for 24 hours, a high-efficiency heterotrophic nitrification-aerobic denitrification strain was screened out according to the results of water quality detection indicators and named Acinetobacter AD-5.

3. The use according to claim 2, characterized in that: In step S1, the formula of the enrichment medium is: 5 g of peptone, 5 g of yeast extract, 10 g of NaCl, and 1 L of distilled water.

4. The use according to claim 2, characterized in that: In step S2, the formula of the acclimation culture medium is: CH3COONa2.93g, KNO3 0.722g, KH2PO4 0.088g, MgSO4·7H2O 0.2g, trace elements 2mL, and distilled water 1L; the trace element formula is: EDTA 15g, ZnSO4 0.2g, MnCl2·4H2O 1.5g, FeSO4·7H2O 0.5g, CuSO4·5H2O 0.5g, CoCl2·6H2O 0.3g, Na2MoO4·2H2O 0.2g, CaCl2 0.1g, and distilled water 1L.

5. The use according to claim 2, characterized in that: In step S3, the BTB culture medium formula is: CH3COONa2.93g, KNO3 0.722g, KH2PO4 0.088g, MgSO4·7H2O 0.2g, trace elements 2mL, 2% agar, 1% BTB, and distilled water 1L; the trace element formula is: EDTA 15g, ZnSO4 0.2g, MnCl2·4H2O 1.5g, FeSO4·7H2O 0.5g, CuSO4·5H2O 0.5g, CoCl2·6H2O 0.3g, Na2MoO4·2H2O 0.2g, CaCl2 0.1g, and distilled water 1L.

6. The use according to claim 2, characterized in that: In step S4, the formula of aerobic nitrification identification medium is: CH3COONa 0.937g, KNO3 0.288g, KH2PO4 0.0351g, MgSO4·7H2O 0.2g, trace elements 2mL, and distilled water 1L; the trace element formula is: EDTA 15g, ZnSO4 0.2g, MnCl2·4H2O 1.5g, FeSO4·7H2O 0.5g, CuSO4·5H2O 0.5g, CoCl2·6H2O 0.3g, Na2MoO4·2H2O 0.2g, CaCl2 0.1g, and distilled water 1L.

7. The use according to claim 2, characterized in that: In step S4, the formula of heterotrophic nitrification identification medium is: CH3COONa 0.937g, NH4Cl 0.153g, KH2PO4 0.0351g, MgSO4·7H2O 0.2g, trace elements 2mL, and distilled water 1L; the trace element formula is: EDTA 15g, ZnSO4 0.2g, MnCl2·4H2O 1.5g, FeSO4·7H2O 0.5g, CuSO4·5H2O 0.5g, CoCl2·6H2O 0.3g, Na2MoO4·2H2O 0.2g, CaCl2 0.1g, and distilled water 1L.

8. A bacterial agent, characterized in that The bacterial agent comprises fermentation broth obtained by fermenting the Acinetobacter AD-5 as described in claim 1.

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