Heterotrophic nitrifying bacteria, method for preparing the same and use thereof

Abstract

The application discloses a heterotrophic nitrifying bacterium and a preparation method and application thereof, and belongs to the technical field of biotechnology.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and more specifically, relates to heterotrophic nitrifying bacteria. This invention also relates to the preparation method and application of the heterotrophic nitrifying bacteria. Background Technology

[0002] Nitrifying bacteria are a type of microorganism widely found in nature. They can convert ammonia nitrogen or nitrite nitrogen in water into other forms of nitrogen through their own metabolism, playing a very important role in the Earth's nitrogen cycle. Since the renowned microbiologist Winogradsky first isolated and purified nitrifying bacteria from soil in 1981, and subsequently proved the mechanism of their nitrification, research on nitrifying bacteria has been continuously carried out.

[0003] As people's living standards improve, the demand for river crabs, shrimp, turtles, and various fish is increasing, driving the rapid development of related aquaculture industries. However, high-density farming and excessive artificial feeding lead to the accumulation of ammonia nitrogen and nitrite in the water. When nitrogen levels in the water exceed the standard, it can cause eutrophication, which can harm the health of farmed organisms and even cause death in severe cases.

[0004] To address this situation, many researchers and probiotic manufacturers both domestically and internationally are dedicated to developing and producing beneficial microorganisms with significant effects and better degradation capabilities, thereby solving the problem of excessive ammonia nitrogen and nitrite levels during the breeding process. Summary of the Invention

[0005] The main objective of this invention is to provide a strain capable of degrading nitrification or denitrification, in order to solve the problem of excessive ammonia nitrogen and nitrite content during aquaculture.

[0006] A heterotrophic nitrifying bacterium, named *Trichosporon asahii*, has been deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on August 8, 2022. The deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] Furthermore, the 26S rRNA sequence of the aforementioned heterotrophic nitrifying bacteria is shown in SEQ ID NO: 1.

[0008] Furthermore, the aforementioned heterotrophic nitrifying bacteria are those that degrade substances with nitrification or denitrification functions.

[0009] The second technical solution provided by this invention is a method for preparing the above-mentioned heterotrophic nitrifying bacteria, which includes the following steps in sequence:

[0010] (1) Sampling: Take an appropriate amount of mud-water mixture from the sewage station in a clean plastic bucket and aerate it with an air pump;

[0011] (2) Take 100ml of water from the aeration tank every 2-3 days to test its ammonia nitrogen, nitrate nitrogen and nitrite nitrogen content;

[0012] (3) Microbial enrichment: After the ammonia nitrogen and nitrite nitrogen in the sample have dropped to below 0.1 mg / L, add NH4Cl standard solution to the container to adjust the NH4 in the sample. + The concentration was increased to 10 mg / L; the ammonia nitrogen index was measured every 2-3 days, and when it dropped to the lowest level, NH4Cl standard solution was added repeatedly to enrich the strains with the ability to degrade ammonia nitrogen.

[0013] (4) Separation and purification: When the number of target bacteria in the sample reaches 10... 5 ~10 8 When the cfu / mL concentration is 1, take 1 mL of the sample and add it evenly to 9 mL of sterile water to make a total of 10. -1 Diluent; and so on, dilute the sample 10 times. 5 ~10 8 The strains were separated from the samples by plate coating method, and the selected strains were preserved.

[0014] The third technical solution provided by this invention is the above-mentioned test method for heterotrophic nitrifying bacteria, which includes the following steps in sequence:

[0015] (1) Culture medium preparation: Prepare the corresponding nitrification, denitrification and LB culture medium according to the culture medium formula, dispense and sterilize for later use;

[0016] (2) Preparation of AF-01 strain: After scraping the prepared slant strain for 20-28 hours, the supernatant was removed by separation, and the sludge was resuspended in an equal amount of pure water for later use.

[0017] (3) Inoculation of test culture medium: Take 5 ml of bacterial sludge resuspension and add it to the nitrification and denitrification culture medium used for the test. There are one aerobic and one relatively anaerobic group. No replicates were set for this test.

[0018] (4) Data measurement: The changes in nitrate nitrogen, ammonia nitrogen and nitrite nitrogen content in the test culture medium were measured according to the test criteria.

[0019] Furthermore, the above-mentioned test method for heterotrophic nitrifying bacteria, the denitrification culture medium preparation method is as follows: 2.0g CH3COONa, 2.0g KNO3, 0.2g MgSO4·7H2O, 1.0g K2HPO4, and 10.0mL of trace element culture medium are added per liter of culture medium, dissolved fully in water, and the pH is adjusted to 7.2. The mixture is then autoclaved at 121℃ for 30min.

[0020] Furthermore, the above-mentioned test method for heterotrophic nitrifying bacteria, the method for preparing the nitrification medium is as follows: 2.0g of CH3COONa, 0.2g of NH4Cl, 0.2g of MgSO4·7H2O, 0.7g of Na2HPO4, 1.0g of KH2PO4, and 10.0mL of trace element medium are added per liter of medium. The medium is then dissolved in water, the pH is adjusted to 7.2, and the medium is autoclaved at 121℃ for 30 minutes.

[0021] Furthermore, the method for preparing the micronutrient culture medium for testing heterotrophic nitrifying bacteria described above is as follows: For each liter of culture medium, add 50.0g of EDTA, 2.2g of ZnSO4, 5.5g of CaCl2, 5.06g of MnCl2·4H2O, 5.0g of FeSO4·7H2O, 1.1g of (NH4)6Mo7O2·4H2O, 1.57g of CuSO4·5H2O, and 1.61g of CoCl2·6H2O. Dissolve these components thoroughly in water, maintain the pH at its natural level, and autoclave at 121℃ for 30 minutes.

[0022] The method for preparing the LB broth culture medium is as follows: Weigh 21g of LB broth culture medium, add water to 1000mL, set the pH to natural, and autoclave at 121℃ for 30min.

[0023] Another technical solution of the present invention is the application of the above-mentioned heterotrophic nitrifying bacteria as a wastewater treatment agent.

[0024] The slow-release wastewater treatment agent containing the above-mentioned heterotrophic nitrifying bacteria comprises the following components by weight percentage: 25-35% porous wood blocks, 0.5-5% sodium alginate solution, 1-5% calcium carbonate solution, and the balance being AF-01 bacterial suspension after resuspension.

[0025] Compared with the prior art, the technical solution provided by the present invention has shown good ability to remove ammonia nitrogen and nitrite nitrogen in high and low ammonia nitrogen concentration test experiments, nitrite removal capacity test experiments and actual application experiments of bacterial sludge. It can be used for various wastewater treatment, especially wastewater treatment in livestock farms. Attached Figure Description

[0026] Figure 1 This is a colony morphology diagram of AF-01;

[0027] Figure 2 This is image A of the microscopic morphology of AF-01;

[0028] Figure 3 This is image B, a microscopic morphology image of AF-01.

[0029] Figure 4 This is the sustained-release curve of AF-01. Detailed Implementation

[0030] The claims of the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0031] Example 1

[0032] This embodiment provides a heterotrophic nitrifying bacterium, deposited at the Guangdong Provincial Microbial Culture Collection Center, with the taxonomic name Trichosporon asahii AF-01, accession number GDMCC No: 62690, deposit date August 8, 2022, and address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0033] Strain identification results:

[0034] Sample AF-01 reproduces asexually through budding, exhibiting pseudohyphae and fungal hyphae, producing arthrospores but no ascospores. After culturing on malt extract agar for 5 days, the colonies are white to grayish-white with a rough, wrinkled surface. See Figure 1 for a colony diagram and Figures 2-3 for magnified views.

[0035] Physicochemical properties are shown in Table 1:

[0036] Table 1

[0037]

[0038]

[0039] 1.3 Molecular identification results

[0040] The 26S rRNA gene sequence obtained from sample AF-01 was 551 bp, and its gene sequence is shown in SEQ ID NO: 1. It was compared with the registered sequence in Genebank using the Blast program; nucleotide homology comparison showed that the 26S rRNA gene sequence of this bacterium was 100% homologous with Trichosporon asahii.

[0041] Example 2

[0042] This embodiment provides a method for preparing the heterotrophic nitrifying bacteria in the embodiment:

[0043] The materials, reagents, and instruments required for the following embodiments are as follows:

[0044] Materials: Cement mixture from the wastewater treatment plant of Boji Pharmaceutical Technology Park

[0045] Reagents and culture media

[0046] (1) Reagents: NaNO2, NH4Cl;

[0047] (2) Culture medium

[0048] LB agar: Weigh 36g of LB agar medium, add water to 1000mL, set pH to normal, and autoclave at 121℃ for 30min.

[0049] (3) Temperature control: room temperature;

[0050] (4) Measurement indicators: nitrate nitrogen, ammonia nitrogen, nitrite nitrogen;

[0051] Its preparation method includes the following steps in sequence:

[0052] (1) Sampling: Take an appropriate amount of mud-water mixture from the sewage treatment plant of Boji Medical Science and Technology Park using a clean plastic bucket and aerate it with an air pump.

[0053] (2) Index detection: Periodically take an appropriate amount of water sample from the aeration tank to test the content of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen.

[0054] (3) Microbial enrichment: After the ammonia nitrogen and nitrite nitrogen in the sample drop to below 0.1 mg / L, add NH4Cl standard solution (c(NH4)) to the container. + ( ) = 1 g / L), adjust the NH4 in the sample + The concentration was reduced to approximately 10 mg / L; the ammonia nitrogen level was measured every 2–3 days, and once it reached its lowest point, NH4Cl standard solution was repeatedly added to enrich the strains capable of degrading ammonia nitrogen.

[0055] (4) Separation and purification: When the number of target bacteria in the sample reaches 10... 5 ~10 8 When the cfu / mL concentration is 1, take a 1 mL sample and add it evenly to 9 mL of sterile water to make a total of 10. -1 Diluent; and so on, dilute the sample to 10. 5 ~10 8 The strains were separated from the samples by plate coating method, and the selected strains were preserved.

[0056] Four bacterial strains with different colony morphologies were screened from the sludge-water mixture of the wastewater treatment plant of Boji Pharmaceutical Technology Park and named JX-F-1, JX-F-2, JX-F-3 and AF-01, respectively.

[0057] Example 3

[0058] Functional testing of AF-01

[0059] The ability of strain AF-01 to degrade ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in nitrified and denitrified media.

[0060] Denitrification medium:

[0061] For each liter of culture medium, add 2.0g of CH3COONa, 2.0g of KNO3, 0.2g of MgSO4·7H2O, 1.0g of K2HPO4, and 10.0mL of trace element culture medium. Dissolve thoroughly in water, adjust the pH to 7.2, and autoclave at 121℃ for 30min.

[0062] Nitrification medium:

[0063] For each liter of culture medium, add 2.0g of CH3COONa, 0.2g of NH4Cl, 0.2g of MgSO4·7H2O, 0.7g of Na2HPO4, 1.0g of KH2PO4, and 10.0mL of trace element culture medium. Dissolve thoroughly in water, adjust the pH to 7.2, and autoclave at 121℃ for 30min.

[0064] Trace element culture medium:

[0065] For each liter of culture medium, add 50.0g EDTA, 2.2g ZnSO4, 5.5g CaCl2, 5.06g MnCl2·4H2O, 5.0g FeSO4·7H2O, 1.1g (NH4)6Mo7O2·4H2O, 1.57g CuSO4·5H2O, and 1.61g CoCl2·6H2O. Dissolve thoroughly in water, maintain natural pH, and autoclave at 121℃ for 30 minutes.

[0066] LB broth culture medium:

[0067] Weigh 21g of LB broth medium, add water to 1000mL, set pH to normal, and autoclave at 121℃ for 30min.

[0068] Test condition control

[0069] (1) Temperature control: Aerobic: 36℃;

[0070] Anaerobic: at room temperature;

[0071] (2) Measurement indicators: nitrate nitrogen, ammonia nitrogen, nitrite nitrogen;

[0072] (3) Testing basis:

[0073] Ammonia nitrogen: Refer to HJ-535-2009 "Determination of Ammonia Nitrogen in Water Quality - Nessler's Reagent Spectrophotometric Method";

[0074] Nitrate nitrogen: Refer to HJ / T346-2007 "Determination of nitrate nitrogen in water quality - ultraviolet spectrophotometry";

[0075] Nitrite nitrogen: Refer to GB7493-87 "Determination of nitrite nitrogen in water - spectrophotometric method".

[0076] Test steps

[0077] (1) Culture medium preparation: Prepare the corresponding nitrification, denitrification and LB culture medium according to the culture medium formula, dispense and sterilize for later use, 100ml per bottle.

[0078] (2) Preparation of AF-01 strain: A small amount of surface bacterial growth was scraped from the prepared slant culture and washed into two 250mL Erlenmeyer flasks containing 100mL LB medium. After incubation at 36℃ and 200rpm for 24h, the culture was centrifuged at 4000rpm for 20 minutes. The supernatant was removed, and the bacterial sludge was resuspended in an equal volume of pure water for later use.

[0079] (3) Inoculation of test culture medium: Take 5 ml of bacterial sludge resuspension and add it to the nitrification and denitrification culture medium used for the test. There are one aerobic and one relatively anaerobic group. No replicates were set for this test.

[0080] (4) Data measurement: The changes in nitrate nitrogen, ammonia nitrogen and nitrite nitrogen content in the test culture medium were measured according to the test criteria.

[0081] Experimental results

[0082] (1) Results of the first functional test are shown in Table 2-5.

[0083] Table 2 Aerobic Denitrification Group

[0084] time Ammonia nitrogen (mg / L) Nitrate (mg / L) Nitrite (mg / L) Total nitrogen oxides (mg / L) Nitrate nitrogen removal rate Total nitrogen removal rate 0h 8.9 144.3 0.0 153.2 24h 2.6 134.5 0.0 137.1 6.79％ 10.51％ 96h 1.3 139.8 0.1 141.1 3.12％ 7.90％ 120h 1.3 138.8 0.1 140.2 3.81％ 8.49％ 144h -0.3 135.8 0.3 135.8 5.89％ 11.36％

[0085] Table 3 Anaerobic Denitrification Group

[0086] time Ammonia nitrogen (mg / L) Nitrate (mg / L) Nitrite (mg / L) Total nitrogen oxides (mg / L) Nitrate nitrogen removal rate Total nitrogen removal rate 0h 9.0 144.9 0.0 153.9 24h 2.3 136.2 0.9 139.5 6.00％ 9.4％ 96h 4.0 71.4 174.7 250.1 50.72％ -62.5％ 120h 6.8 67.3 168.9 242.9 53.55％ -57.8％ 144h 4.3 64.2 176.6 245.1 55.69％ -59.3％

[0087] Table 4 Aerobic Nitrification Group

[0088] time (ammonia nitrogen mg / L) Nitrate (mg / L) Nitrite (mg / L) Total nitrogen oxides (mg / L) ammonia nitrogen removal rate Total nitrogen removal rate 0h 60.8 -0.3 0.0 60.5 24h 43.7 -2.0 0.0 41.7 28.13％ 31.07％ 96h 48.4 -1.2 0.0 47.2 20.39％ 21.98％ 120h 51.4 -2.1 0.0 49.4 15.46％ 18.35％ 144h 48.4 -1.0 0.1 47.6 20.39％ 21.32％

[0089] Table 5 Anaerobic Nitrification Group

[0090] time (ammonia nitrogen mg / L) Nitrate (mg / L) Nitrite (mg / L) Total nitrogen oxides (mg / L) ammonia nitrogen removal rate Total nitrogen removal rate 0h 42.8 0.1 0.0 42.9 24h 20.0 -1.0 0.3 19.2 53.27％ 55.24％ 96h 18.2 -1.5 0.0 16.7 57.48％ 61.07％ 120h 21.4 -2.2 0.0 19.3 50.00％ 55.01％ 144h 16.9 -0.9 0.0 16.0 60.51％ 62.70％

[0091] (2) Secondary verification experiment, see Table 6-9.

[0092] Table 6 Aerobic Denitrification Group

[0093] time Ammonia nitrogen (mg / L) Nitrate (mg / L) Nitrite (mg / L) Total nitrogen oxides (mg / L) Nitrate nitrogen removal rate Total nitrogen removal rate 0h 6.4 140.2 0.0 146.7 16h 0.91 137.82 0.04 138.77 1.70％ 5.41％ 40h -0.3 38.7 0.1 38.5 / / 64h 0.8 132.3 0.2 133.3 5.63％ 9.13％

[0094] Table 7 Anaerobic Denitrification Group

[0095] time Ammonia nitrogen (mg / L) Nitrate (mg / L) Nitrite (mg / L) Total nitrogen oxides (mg / L) Nitrate nitrogen removal rate Total nitrogen removal rate 0h 6.4 124.3 0.0 130.7 16h 1.43 136.26 0.79 138.49 -9.62％ -5.96％ 40h 0.9 39.2 97.2 137.3 68.46％ -5.05％ 64h 3.8 62.1 159.4 225.3 50.04％ -72.38％

[0096] Table 8 Aerobic Nitrification Group

[0097] time Ammonia nitrogen (mg / L) Nitrate (mg / L) Nitrite (mg / L) Total nitrogen oxides (mg / L) ammonia nitrogen removal rate Total nitrogen removal rate 0h 48.4 0.5 0.0 48.9 16h 15.49 -1.18 0.05 14.35 68.00％ 70.65％ 40h 17.7 -0.4 0.0 17.3 63.43％ 64.62％

[0098] Table 9 Anaerobic Nitrification Group

[0099]

[0100]

[0101] (3) Results of low-concentration ammonia nitrogen test, the results are shown in Table 10-13;

[0102] Table 10 Ammonia Nitrogen Test Groups at 10 mg / L Concentration

[0103] time Ammonia nitrogen (mg / L) Nitrate (mg / L) Nitrite (mg / L) Total nitrogen oxides (mg / L) ammonia nitrogen removal rate 0h 11.58 -1.11 0.01 11.59 20h 1.68 / 0.03 1.71 85.49％

[0104] Table 11 5 mg / L ammonia nitrogen concentration test group

[0105] time Ammonia nitrogen (mg / L) Nitrate (mg / L) Nitrite (mg / L) Total nitrogen oxides (mg / L) ammonia nitrogen removal rate 0h 6.68 1.36 0.01 11.37 16h 0.56 -1.46 0.01 0.57 91.62％

[0106] (4) Test results of nitrite nitrogen degradation effect

[0107] Table 11 Nitrite Blank Control

[0108] time ammonia nitrogen nitrates nitrite 24h 0.675 3.158 9.371 40h / / 7.465 60h / / 7.723

[0109] Table 12 Nitrite Degradation Effect Test

[0110] time Ammonia nitrogen (mg / L) Nitrate (mg / L) Nitrite (mg / L) Total nitrogen oxides (mg / L) Nitrite nitrogen removal rate 0h / -0.47 11.13 11.13 20h / -0.52 10.76 10.76 3.26％ 44h / -0.25 0.002 0 99.98％ 60h / / 0.008 0.008 99.93％

[0111] Note: The 60h data set is the validation data for the 44h data set.

[0112] Table 13 Secondary verification of nitrite degradation effect

[0113]

[0114]

[0115] The above experiments show that AF-01 achieved a maximum anaerobic nitrification ammonia nitrogen removal rate of 69.27% ​​in a high-concentration ammonia nitrogen test medium after 40 hours. Based on the results of two measurements, its ability to reduce ammonia nitrogen under relatively anaerobic conditions is slightly better. Under aerobic conditions with an initial ammonia nitrogen concentration of 11.58 mg / L, aerobic nitrification is strong, reducing the ammonia nitrogen concentration to 1.68 mg / L after 20 hours with a degradation rate of 85.49% and no nitrite production. Under aerobic conditions with an initial ammonia nitrogen concentration of 6.68 mg / L, aerobic nitrification is also strong, reducing the ammonia nitrogen concentration to 0.56 mg / L after 16 hours with a degradation rate as high as 91.62%, and it does not produce other forms of nitrogen salts while reducing ammonia nitrogen.

[0116] In the denitrification test medium, the strain exhibited a strong ability to reduce nitrate nitrogen under anaerobic conditions, but the conversion process was incomplete, primarily involving the conversion of nitrate nitrogen to nitrite nitrogen. In the nitrite nitrogen degradation test, under aerobic conditions, with an initial concentration of 11 mg / L, the nitrite concentration decreased to 0.002 mg / L after 44 hours, achieving a degradation rate of 99.98%. In the secondary verification experiment, under aerobic conditions, with an initial concentration of 10 mg / L, the nitrite nitrogen removal rate reached 99.90% after 36 hours. This indicates that the strain has a good ability to remove nitrite nitrogen.

[0117] In summary, regarding the nitrification of strain AF-01: under high concentration (40 mg / L) ammonia nitrogen conditions, anaerobic nitrification is slightly better than aerobic nitrification; under low concentration (around 10 mg / L) ammonia nitrogen conditions, aerobic nitrification is significant; this strain does not produce other forms of nitrogen salts during nitrification.

[0118] Example 4

[0119] AF-01 microbial sludge practical application experiment

[0120] The effect of strain AF-01 on the removal of ammonia nitrogen in wastewater from Guanghe Electronics Factory.

[0121] Experimental samples

[0122] (1) Experimental bacterial agent: AF-01

[0123] (2) Experimental water sample:

[0124] Raw water sample from the wastewater treatment plant of Guanghe Electronics Factory;

[0125] Water sample from the end of the aerobic tank at the wastewater treatment plant of Guanghe Electronics Factory.

[0126] Experimental condition control

[0127] 1. Temperature control: Aerobic, 36℃

[0128] 2. Measurement indicators: nitrate nitrogen, ammonia nitrogen, nitrite nitrogen;

[0129] 3. Testing basis:

[0130] Ammonia nitrogen: Refer to HJ-535-2009 "Determination of Ammonia Nitrogen in Water Quality - Nessler's Reagent Spectrophotometric Method";

[0131] Nitrate nitrogen: Refer to HJ / T346-2007 "Determination of nitrate nitrogen in water quality - ultraviolet spectrophotometry";

[0132] Nitrite nitrogen: Refer to GB7493-87 "Determination of nitrite nitrogen in water - spectrophotometric method".

[0133] Experimental steps

[0134] 1. Water quality testing: First, the water sample is tested for initial indicators.

[0135] 2. Sample preparation:

[0136] (1) Adjust the pH of the raw water of Guanghe Electronics Factory sewage treatment plant to 10, add ZnSO and stir evenly to flocculate and precipitate, then filter and adjust the pH of the supernatant to 7.5, and put it into 250mL Erlenmeyer flasks, 100ml in each flask.

[0137] (2) Adjust the pH of the water sample at the end of the aerobic tank of the wastewater treatment plant of Guanghe Electronics Factory to 7-8, add ZnSO and stir evenly to flocculate and precipitate, then filter and adjust the pH of the supernatant to 7.5, and put it into 250mL Erlenmeyer flasks, 100ml in each flask.

[0138] 3. Add experimental bacterial agent: Add experimental bacterial agent AF-01 to the above-treated samples and the original water sample at the end of the aerobic tank, respectively, at a dosage of 5%; the experimental design is shown in Table 14 below.

[0139] Table 3-13 Experimental Design for Practical Application of Mushroom Substrate

[0140]

[0141] 4. Test Results

[0142] (1) The test results of the original solution are shown in Table 15 below.

[0143] Table 15 Results of the original solution test

[0144] Serial Number design 0h 24h Removal rate 1 After flocculation, the original solution is shaken on a shaker. 90.3 mg / L 72.0 mg / L 20％ 2 After flocculation, the original solution + (after adding the bacterial inoculum and shaking well) was allowed to stand. 90.3 mg / L 73.9 mg / L 18％

[0145] (2) The test results at the end of the aerobic tank are shown in Table 16 below.

[0146] Table 16 Results of end-of-pipe tests in the aerobic tank

[0147]

[0148]

[0149] Through the above experiments, it can be found that in typical electronic wastewater with low BOD and high COD, under aerobic conditions, the experimental strain AF-01 achieved the highest ammonia nitrogen removal rate of 20% in the raw water of Guanghe Electronics Factory wastewater treatment plant at 24 hours, reducing the ammonia nitrogen in the raw wastewater from 90.3 mg / L to 72 mg / L. Based on the results of the two measurements, its ability to reduce ammonia nitrogen under aerobic conditions was slightly better. In the water sample at the end of the aerobic tank, under aerobic conditions, the highest ammonia nitrogen removal rate reached 31.2% at 48 hours, reducing the ammonia nitrogen in the water sample at the end of the aerobic tank from 51.8 mg / L to 36.5 mg / L.

[0150] In tests with high and low ammonia nitrogen concentrations, tests on nitrite removal capacity, and practical applications of the bacterial sludge, it was found that strain AF-01, selected from the sludge-water mixture of the Boji Pharmaceutical Science and Technology Park wastewater treatment plant, has a relatively good ability to remove ammonia nitrogen and nitrite nitrogen.

[0151] Example 5: Production of AF-01 sustained-release products

[0152] Materials and Equipment

[0153] Materials: porous wood blocks, resuspended AF-01 bacterial culture, sodium alginate solution (0.5-5%), calcium carbonate solution (1-5%)

[0154] Facilities and equipment: autoclave, negative pressure device, centrifuge

[0155] Preparation method:

[0156] Preliminary preparation: After centrifuging the cultured AF-01 bacterial culture, remove the supernatant and resuspend it in sterile water to twice the original concentration;

[0157] Soak the porous wood blocks in salt water of 3 times their weight, then autoclave them, cool them, and drain off the moisture for later use.

[0158] After the wood blocks have been drained, they are added to the resuspended bacterial solution, placed in a negative pressure device and soaked for 24 hours. Then, they are taken out and drained of surface moisture.

[0159] Add sodium alginate solution to the wood block and stir well, then add it to the calcium carbonate solution;

[0160] After fixing for 2 hours, remove and drain to form a slow-release product.

[0161] Product performance testing

[0162] Wood block absorption performance test

[0163] Experimental steps

[0164] After weighing the wood blocks, soak them completely in water and then sterilize them in an autoclave.

[0165] Sterilization conditions: 121℃, 30min, as shown in Table 17 below.

[0166] Table 17

[0167]

[0168]

[0169] The weight of a wooden block increases by approximately twice its original weight after autoclaving.

[0170] Product sustained-release profile development

[0171] Experimental materials

[0172] (1) Physiological saline: 0.85% physiological saline

[0173] (2) Culture medium:

[0174] LB agar: Weigh 36g of LB agar medium, add water to 1000mL, set pH to normal, and autoclave at 121℃ for 30min.

[0175] Experimental steps:

[0176] (1) Prepare the required culture medium and physiological saline, and sterilize them for later use.

[0177] (2) Add 1% of the microbial material to 1L of physiological saline. Take samples from the physiological saline in the beaker every 3 hours to determine the total number of microorganisms. Sampling times are 8:00 AM, 11:00 AM, 2:00 PM, 5:00 PM, 8:00 AM the following morning, and 8:00 AM the day after that. At the same time, retain all water samples and test the total number of bacteria in the samples during the next sampling.

[0178] (2) Total bacterial count determination: Five dilution gradients were used for counting, with two replicates for each gradient. The plate count method was used, and the mold incubator was inverted at 36 degrees Celsius for 48 hours.

[0179] (3) Record and calculate the number of live bacteria released by the product at each time period, and create a sustained-release curve. (Refer to...) Figure 4 .

[0180] Experimental results

[0181] (1) The number of viable bacteria in samples taken at each time period is recorded in Table 18 below.

[0182] Table 18 Viable bacteria count in samples taken at different time periods

[0183]

[0184] Product long-lasting release experiment

[0185] 1. Experimental Objective: To determine the efficiency of long-term microbial release from microbial materials.

[0186] 2. Experimental equipment: autoclave, mold incubator, clean bench;

[0187] 3. Experimental Materials

[0188] (1) Physiological saline: 0.85% physiological saline

[0189] (2) Culture medium:

[0190] LB agar: Weigh 36g of LB agar medium, add water to 1000mL, set pH to normal, and autoclave at 121℃ for 30min.

[0191] 3. Experimental steps:

[0192] (1) Prepare the required culture medium and physiological saline, and sterilize them for later use.

[0193] (2) Add 1% of the microbial material to 1L of physiological saline. After 24 hours, measure the total number of colonies in the physiological saline. Discard the physiological saline in the beaker, add 1L of physiological saline to the beaker, and measure the total number of colonies in the physiological saline in the beaker after 24 hours. The counting is performed using 4 dilution gradients. Repeat the above operation for 4 days.

[0194] (3) Total bacterial count determination: After incubation in an upside-down mold incubator at 36 degrees Celsius for 48 hours, the number of viable bacteria in physiological saline was determined by plate counting method.

[0195] 4. The experimental results are shown in Table 19 below.

[0196] Table 19 Viable bacteria count every 24 hours

[0197] Time (days) Bacterial count (cfu / ml) 1 <![CDATA[6.5×10 7 ]]> 2 <![CDATA[8.5×10 7 ]]> 3 <![CDATA[1.59×10 8 ]]> 4 <![CDATA[7.9×10 7 ]]>

[0198] This demonstrates that the microbial material provided in this application has the effect of releasing microorganisms over a long period of time.

Claims

1. A heterotrophic bacterium that degrades ammonia nitrogen and nitrite nitrogen. AF-01 It is deposited at the Guangdong Provincial Center for Microbial Culture Collection, and its taxonomic name is Trichosporon asahii Accession number: GDMCC No: 62690; accession date: August 8, 2022.

2. The heterotrophic bacteria for degrading ammonia nitrogen and nitrite nitrogen as described in claim 1 AF-01 The test method is characterized by, The steps are as follows: (1) Culture medium preparation: Prepare the corresponding nitrification, denitrification and LB culture medium according to the culture medium formula, dispense and sterilize for later use; (2) Preparation of AF-01 strain: A small amount of surface bacterial growth was scraped from the prepared slant strain and washed into a 250mL Erlenmeyer flask containing 100mL LB medium. After culturing at 36℃ and 200rpm for 24h, the culture was centrifuged at 4000rpm for 20 minutes. After removing the supernatant, the bacterial sludge was resuspended in an equal volume of pure water for later use. (3) Inoculation of test culture medium: Take 5 ml of bacterial sludge resuspension and add it to the nitrification and denitrification culture medium used for the test. There are one aerobic and one relatively anaerobic group. No replicates were set for this test. (4) Data measurement: The changes in nitrate nitrogen, ammonia nitrogen and nitrite nitrogen content in the test culture medium were measured according to the test criteria; The denitrification medium is prepared as follows: 2.0g of CH3COONa, 2.0g of KNO3, 0.2g of MgSO4·7H2O, 1.0g of K2HPO4, and 10.0mL of trace element medium are added per liter of medium. The medium is dissolved in water, the pH is adjusted to 7.2, and the medium is autoclaved at 121℃ for 30min. The nitrification medium is prepared as follows: 2.0g of CH3COONa, 0.2g of NH4Cl, 0.2g of MgSO4·7H2O, 0.7g of Na2HPO4, 1.0g of KH2PO4, and 10.0mL of trace element medium are added per liter of medium. The medium is dissolved in water, the pH is adjusted to 7.2, and the medium is autoclaved at 121℃ for 30min. The LB medium preparation method is as follows: Weigh 21g of LB broth medium, add water to 1000mL, set the pH to natural, and autoclave at 121℃ for 30min.

3. The heterotrophic bacteria for degrading ammonia nitrogen and nitrite nitrogen according to claim 2 AF-01 The test method is characterized by, The method for preparing the trace element culture medium is as follows: 50.0g of EDTA, 2.2g of ZnSO4, 5.5g of CaCl2, 5.06g of MnCl2·4H2O, 5.0g of FeSO4·7H2O, 1.1g of (NH4)6Mo7O2·4H2O, 1.57g of CuSO4·5H2O, and 1.61g of CoCl2·6H2O are added per liter of culture medium. The medium is then dissolved in water, the pH is left to stand, and the medium is autoclaved at 121℃ for 30 minutes.

4. The heterotrophic bacteria for degrading ammonia nitrogen and nitrite nitrogen as described in claim 1 AF-01 Application as a treatment agent for removing ammonia nitrogen from wastewater.

5. A heterotrophic bacterium for degrading ammonia nitrogen and nitrite nitrogen as described in claim 1. AF-01 A slow-release ammonia nitrogen treatment agent for wastewater, characterized in that, The components include the following weight percentages: 25-35% porous wood blocks, 0.5-5% sodium alginate solution, 1-5% calcium carbonate solution, and the balance being AF-01 bacterial suspension after resuspension.

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