Pseudomonas tai H1R1 and application thereof in treatment of mariculture tail water

Abstract

The invention discloses Pseudomonas tai H1R1 and application thereof in treatment of mariculture tail water, the Pseudomonas tai H1R1 has excellent high salt resistance and aerobic denitrification function, can directly convert ammonia nitrogen and nitrate into nitrogen and efficiently remove nitrogen, the nitrogen removal rate can reach 80% or above, nitrogen load in water is reduced, the nitrogen removal efficiency is improved, and the application of the Pseudomonas tai H1R1 in treatment of mariculture tail water is improved. The problem of water eutrophication caused by nitrogen can be prevented; the system can survive and breed under various severe environment conditions and treat mariculture tail water. The pseudomonas tai H1R1 disclosed by the invention can utilize organic matters in mariculture tail water as a carbon source, so that the growth speed and the growth amount of bacteria are increased, and organic wastes in a culture process are reduced, so that the strain H1R1 can promote self growth while treating the mariculture tail water; and double benefits of environmental governance and resource cyclic utilization are realized.

Description

(I)TECHNICAL FIELD

[0001] The present application belongs to the technical field of wastewater treatment, and specifically relates to a new strain of salt-tolerant aerobic denitrifying bacteria, Pseudomonas taishanensis H1R1, and application thereof in treating seawater aquaculture tail water. (II)BACKGROUND

[0002] As a large country of seawater aquaculture, the output of seawater aquaculture in China has steadily increased year by year, from 35,858,200 tons in 2023 to 36,652,900 tons in 2024, with an increase of 2.22%. At the same time, the area of seawater with eutrophication in summer in 2023 was 28,960 square kilometers, an increase of 190 square kilometers compared with the previous year. The main over-standard index in the water body of the key seawater aquaculture area is inorganic nitrogen. The proportions of the areas with inorganic nitrogen, active phosphate, and chemical oxygen demand in the water body exceeding the evaluation standard are 54.2%, 71.8%, and 98.6%, respectively, of the monitored area. Therefore, the environmental problems caused by the discharge of seawater aquaculture tail water have attracted increasing attention. The pollutants in seawater aquaculture tail water contain undigested leftover feed, fish medicines, and metabolic waste of cultured organisms, among which excessive nutrients such as nitrogen and phosphorus can lead to water eutrophication and cause excessive proliferation of algae. With the emphasis of the country on environmental protection, relevant regulations and policies have put forward increasingly strict requirements for the discharge of seawater aquaculture tail water. The seawater aquaculture industry must strengthen the treatment of tail water.

[0003] Patent application CN116062904B discloses a method for treating wastewater and plant growth promotion by using a heterotrophic nitrification-aerobic denitrification bacterium. The heterotrophic nitrification-aerobic denitrification bacterium in the method grows fast under the conditions of sodium acetate as carbon source, carbon-nitrogen ratio of 10, pH value of 7, culture temperature of 30-40°C, and rotation speed of 180 r / min. However, the removal effect of the above-mentioned strain on different valence nitrogen sources is different, and the removal rate of nitrite nitrogen is relatively low, only 18.53%. Patent application CN119372112A discloses a method for treating aquaculture wastewater, pharmaceutical wastewater, and other water bodies by using aerobic denitrifying Halomonas halocenarum LHT6. The aerobic denitrifying Halomonas halocenarum LHT6 in the method can grow and remove nitrate under the conditions of carbon-nitrogen ratio of 2-10, pH value of 7-11, and 0%-15% NaCl. The strain not only tolerates multiple antibiotics, but also can treat nitrate in water bodies containing various heavy ions. However, the treatment effect of the strain may be affected when treating seawater aquaculture tail water and other water bodies with high salt content.

[0004] Therefore, there is a need to screen a salt-tolerant aerobic denitrifying bacterium for treating seawater aquaculture tail water. (III)SUMMARY

[0005] The application aims to provide Pseudomonas tiananensis H1R1 and application thereof in treating mariculture tail water, the Pseudomonas tiananensis H1R1 belongs to a salt-tolerant aerobic denitrifying bacterium, can survive and reproduce under various harsh environmental conditions, and can directly convert ammonia nitrogen and nitrate into nitrogen, thereby efficiently removing nitrogen and effectively controlling the total nitrogen content in mariculture tail water, and solving the problem of high nitrogen content in the existing mariculture tail water.

[0006] The technical solution adopted by the application is as follows:

[0007] The application provides a new salt-tolerant aerobic denitrifying bacterial strain, Pseudomonas tiananensis H1R1, which is preserved in the China General Microbiological Culture Collection Center, has a preservation number of CGMCC No. 34390, a preservation date of April 28, 2025, and a preservation address of No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road 1st Courtyard.

[0008] The Pseudomonas tiananensis H1R1 is a salt-tolerant aerobic denitrifying bacterium screened from the sediment of a mariculture tail water discharge ditch of a certain South American white shrimp breeding factory in Dinghai District, Zhoushan City, Zhejiang Province, has a nearly round colony morphology, presents white color, has a raised center, has uniform and shiny surface texture, and has a clear and regular boundary zone, and shows rod-shaped cells under an electron microscope.

[0009] The application also provides application of the Pseudomonas tiananensis H1R1 in treating mariculture tail water, and the application is to reduce the total nitrogen content in mariculture tail water.

[0010] Further, the method of the application is as follows: (1) inoculating the Pseudomonas tiananensis H1R1 into a liquid LB enrichment culture medium, enriching and culturing under the condition of 20-30 DEG C and 100-150 r / min to obtain a bacterial liquid; the liquid LB enrichment culture medium is composed of 10 g / L of tryptone, 5 g / L of yeast extract, 0.1 g / L of KNO3, 35 g / L of NaCl, and water as a solvent, and has a pH of 7; (2) inoculating the bacterial liquid of step (1) into mariculture tail water, stirring at 20-25 DEG C and 130-150 r / min until the total nitrogen removal rate is higher than 80%, and discharging the mariculture tail water.

[0011] Further, the enrichment and culturing condition in step (1) is to enrich and culture under the condition of 25 DEG C and 150 r / min for 48 h.

[0012] Further, the OD of the bacterial liquid in step (1) is 0.5-1.3, preferably 0.8. 600

[0013] ​Further, the step (2) inoculates the seawater breeding tail water with the bacteria solution in an amount of 1-5% by volume concentration, preferably 2% by volume concentration.

[0014] Further, the seawater breeding tail water has a NaCl content of 10-100 g / L (preferably 35 g / L), an amino nitrogen (NH4 + -N) content of 1-10 mg / L (preferably 2 mg / L), and a nitrate nitrogen (NO3 - -N) content of 10-30 mg / L (preferably 10 mg / L).

[0015] Further, the seawater breeding tail water is simulated seawater breeding tail water with a final concentration composition of KNO3 0.07215 g / L, C4H6O4 (succinic acid) 0.1688 g / L, KH2PO4 0.00527 g / L, NH4Cl 0.00764 g / L, NaHCO3 0.05 g / L, NaCl 35.0 g / L, and deionized water as the solvent.

[0016] Compared with the prior art, the beneficial effects of the present application mainly include:

[0017] (1) The Pseudomonas taishanensis H1R1 of the present application has excellent aerobic denitrification function, can directly convert ammonia nitrogen and nitrate into nitrogen, has high nitrogen removal efficiency, and the denitrification rate can reach more than 80%, which not only reduces the nitrogen load in the water body, but also helps to prevent the eutrophication problem caused by nitrogen in the water body.

[0018] (2) The Pseudomonas taishanensis H1R1 of the present application can adapt to different salinity, so that it can survive and reproduce in various harsh environmental conditions and treat seawater breeding tail water.

[0019] (3) The Pseudomonas taishanensis H1R1 of the present application can utilize the organic matter in the seawater breeding tail water as a carbon source, which not only improves the growth rate and amount of the bacteria, but also helps to reduce the organic waste during the breeding process, so that the strain H1R1 can promote its own growth while treating the seawater breeding tail water, realizing the dual benefits of environmental governance and resource recycling. (Four) Description of Drawings

[0020] Figure 1 It is a scanning electron microscope image of the strain H1R1.

[0021] Figure 2 It is a phylogenetic tree of the strain H1R1.

[0022] Figure 3 It is a curve graph of the content of total nitrogen (TN) and nitrate nitrogen (NO3 - -N) in the simulated seawater breeding wastewater treated by the strain H1R1 changing with time.

[0023] Figure 4 Figure 1 is a graph of the growth amount of strain H1R1 and its removal rate of total nitrogen and nitrate nitrogen in simulated mariculture wastewater.

[0024] Figure 5 Figure 2 is a graph of the OD value of strain H1R1 in treating simulated mariculture wastewater with different salinity.

[0025] Figure 6 Figure 3 is a graph of the nitrogen removal rate and OD value of strain H1R1 in treating actual mariculture tail water in Example 4. (V) DETAILED DESCRIPTION

[0026] The application will be further described in conjunction with specific examples, but the protection scope of the application is not limited to this:

[0027] The room temperature in the embodiments of the application refers to 25-30℃.

[0028] The medium composition in the embodiments of the application is as follows:

[0029] The liquid BTB medium is composed of KNO3 1.0 g / L, C4H6O4 (succinic acid) 1.0 g / L, KH2PO4 1.0 g / L, CaCl2 0.5 mL / L, MgSO4·7H2O 0.5 mL / L, 1% bromothymol blue 1 mL / L, NaOH 0.2 g / L, NaCl 35.0 g / L, trace element solution 100 μL / L, deionized water as solvent, and pH 7.0; the trace element solution is composed of EDTA 50.0 g / L, ZnSO4 2.2 g / L, CaCl2 5.5 g / L, MnCl2·4H2O 2.06 g / L, FeSO4·7H2O 5.0 g / L, (NH4)6M7O2·7H2O 1.1 g / L, CuSO4·5H2O 1.57 g / L, CoCl2·6H2O 1.61 g / L, deionized water as solvent, and pH 6.0.

[0030] The solid BTB medium is obtained by adding agar 20.0 g / L to the liquid BTB medium.

[0031] The liquid LB enrichment medium is composed of tryptone 10 g / L, yeast extract 5 g / L, KNO3 0.1 g / L, NaCl 35 g / L, deionized water as solvent, and pH 7.0. The solid LB enrichment medium is obtained by adding agar 20.0 g / L to the liquid LB enrichment medium.

[0032] The total nitrogen (TN) is detected by alkaline potassium persulfate digestion-ultraviolet spectrophotometry (referring to the national standard method HJ636-2012).

[0033] Ammonia nitrogen (NH4 + -N) was detected by using the reagent spectrophotometric method (refer to HJ 535-2009).

[0034] Nitrate nitrogen (NO3 - -N) was detected by using the ultraviolet spectrophotometric method (refer to HJ / T 346-2007).

[0035] Nitrite nitrogen (NO2 - -N) accumulation was detected by using the molecular absorption spectrophotometric method (refer to GB 7493-87).

[0036] Example 1, Isolation and identification of strain H1R1

[0037] 1. Isolation of strain H1R1

[0038] (1) Strain source

[0039] Strain H1R1 was derived from the sediment of a seawater aquaculture tail water discharge ditch of a Penaeus vannamei aquaculture farm in Dinghai District, Zhoushan City, Zhejiang Province.

[0040] (2) Enrichment of the strain

[0041] 5.0 g of the sediment was taken into a conical flask containing 50 mL of sterile water, and after standing at room temperature for a period of time, 10 mL of the supernatant was mixed with 90 mL of liquid LB enrichment medium, and the culture was carried out at a rotation speed of 130 r / min and a temperature of 25℃ for 3 days. If the liquid is clear, the culture time is extended; if the liquid is turbid, continue to take 10 mL of the supernatant and mix it with 90 mL of liquid LB enrichment medium for culture, and repeat the enrichment experiment three times to improve the concentration of the bacterial solution.

[0042] (3) Isolation and purification of the strain

[0043] The bacterial solution obtained after enrichment was diluted with sterile water in a gradient of 10 -1 to 10 -9 , 1 mL of the 10 -4 to 10 -6 gradient dilution was taken and spread on a solid BTB medium plate, and placed in a constant temperature incubator at 25℃ for 3 days of standing culture until colonies were formed. Then, the colonies in the blue area were selected and separated by repeated streaking on a new solid BTB medium plate, and the streaking separation and purification were repeated three times to obtain the purified strain, which was preserved and numbered.

[0044] (4) Screening of the strain

[0045] The screened strains were inoculated into new solid LB enrichment medium and incubated at 25°C for 48h. Then they were inoculated into liquid BTB medium and incubated at 25°C, 130r / min for 72h. The supernatant was centrifuged and the growth (OD 600 ) and NH4 + -N removal rate, NO2 - -N accumulation, NO3 - -N removal rate of the supernatant were measured, as shown in Table 1. The strain with good biomass (OD 600 >0.6) and the best nitrogen removal rate was selected as the dominant strain, and was recorded as strain H1R1.

[0046] Table 1 Performance parameters of some screened strains

[0047]

[0048]

[0049] 2. Identification of strain H1R1

[0050] (1) Morphological characteristics of strain H1R1

[0051] Strain H1R1 was inoculated into solid LB enrichment medium and incubated at 25°C for 24h. The colony morphology showed a nearly round profile, with white color, and the central part of the colony was raised. The surface texture was uniform and had a moist feeling, and the edge area formed a clear and regular boundary. Under an electron microscope, the cells were rod-shaped (as shown in Figure 1 ).

[0052] (2) 16S rDNA identification of strain H1R1

[0053] The purified strain H1R1 was sent to Beijing Xindai Zhonghe Technology Co., Ltd. for 16S rDNA sequencing (shown in SEQ ID NO. 1). Through BLAST comparison in NCBI database, the sequence showed 100% similarity with Pseudomonas sp. strain C-S-R1-17 and Pseudomonas taeanensis strain JP09. Using MEGA11.0 to construct a neighbor-joining phylogenetic tree ( Figure 2 ), it was confirmed that strain H1R1 belongs to the genus Pseudomonas, and was named Pseudomonas taeanensis H1R1. It was preserved in the China General Microbiological Culture Collection Center, with the preservation number CGMCC No. 34390, the preservation date April 28, 2025, and the address No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road No. 1.

[0054] The 16S rDNA sequence of H1R1 is as follows:

[0055] TAACACATGCAAGTCGAGCGGTAGAGAGGAGCTTGCTTCTCTTGAGAGCGGCGGACGG

[0056] GTGAGTAATGCCTAGGAATCTGCCTAGTGGTGGGGGATAACGTTCGGAAACGGACGCTAAT

[0057] ACCGCATACGTCCTACGGGAGAAAGCGGGGGATCTTCGGACCTCGCGCCATTAGATGAGCC

[0058] TAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGTAACTGGTC

[0059] TGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGC

[0060] AGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAA

[0061] GGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGTTGTAGATTAATACTCTGCAA

[0062] TTTTGACGTTACCGACAGAATAAGCACCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACG

[0063] AAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTCGTTAAGT

[0064] TGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCGAGCTAGAGTA

[0065] CGGTAGAGGGTAGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACA

[0066] CCAGTGGCGAAGGCGACTACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAG

[0067] CAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGAAT

[0068] CCTTGAGATTTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCA

[0069] AGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATT

[0070] CGAAGCAACGCGAAGAACCTTACCTGGCCTTGACATGCTGAGAACTTTCCAGAGATGGATT

[0071] GGTGCCTTCGGGAACTCAGACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGAT

[0072] GTTGGGTTAAGTCCCGTAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGG

[0073] GCACTCTAAGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATC

[0074] ATGGCCCTTACGGCCAGGGCTACACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGC

[0075] CGCGAGGTGGAGCTAATCCCATAAAACCGATCGTAGTCCGGATCGCAGTCTGCAACTCGAC

[0076] TGCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGG

[0077] GCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCTCCAGAAGTAGCTAGTCTAA

[0078] CCTTCGGGG.

[0079] Example 2: Denitrification function of strain H1R1

[0080] (1) Bacterial solution

[0081] The H1R1 strain on the plate was picked up with an inoculation loop and inoculated into liquid LB enrichment medium for activation culture. It was cultured at 25℃ and 130 rpm for 48 h to obtain OD. 600 The bacterial solution has a concentration of 0.8.

[0082] (2) Denitrification function

[0083] The final concentration composition (g / L) of simulated seawater aquaculture tailwater was as follows: KNO3 0.07215, C4H6O4 (succinic acid) 0.1688, KH2PO4 0.00527, NH4Cl 0.00764, NaHCO3 0.05, NaCl 35.0. The pH was adjusted to 7, and the solvent was deionized water. The amino nitrogen content was 2 mg / L, and the nitrate nitrogen content was 10 mg / L.

[0084] The microbial preparation was purchased from Yangzhou Haicheng Biotechnology Co., Ltd., and the product name is denitrifying bacteria agent.

[0085] Three experimental groups were set up: an experimental group, a control group, and a positive control group, with three parallel experiments in each group. Equal volumes of simulated seawater aquaculture wastewater were added to all three groups. The experimental group received 2% (v / v) of the bacterial solution prepared in step (1), the positive control group received 2% (v / v) of the microbial agent, and the control group received no treatment. Denitrification was performed at 25℃ and 130 r / min, and OD values ​​were measured at 0h, 8h, 16h, 24h, 32h, and 40h. 600 Values ​​and total nitrogen (TN) and nitrate nitrogen (NO3) in water bodies - -N) content, results are shown in Figure 3 Removal rates of various substances and OD values ​​of strains within 40 hours 600 Worth seeing Figure 4 The results showed that by 40 hours, the nitrate nitrogen removal rate of strain H1R1 reached 82%, the total nitrogen removal rate reached 80%, and the OD... 600 The concentration reached 0.83, indicating good growth. The nitrate nitrogen removal rate in the positive control group was 68%, and the total nitrogen removal rate was 63.33%.

[0086] Example 3: Salt tolerance of strain H1R1

[0087] (1) Bacterial solution

[0088] Strain H1R1 was inoculated into liquid LB enrichment medium, and enriched at 25℃, 130r / min for 48h to obtain OD 600 value of 0.8.

[0089] (2) Salt tolerance

[0090] The formula of simulated marine aquaculture tail water was the same as that of Example 2.

[0091] Five groups of simulated marine aquaculture tail water with different salinity gradients were set up, and the salinity was 15g / L, 35g / L, 55g / L, 75g / L and 95g / L. Each group of experiment was added with 2% of the bacterial liquid prepared in step (1) by volume concentration, and the OD 600 value of the bacterial liquid was measured after 24h of treatment at 25℃, 130r / min. The results are shown in Table 3. Figure 5 The results showed that the H1R1 strain could tolerate salinity of 15-75g / L.

[0092] Example 4: Practical application of strain H1R1 in treating marine aquaculture tail water

[0093] 3L of water was taken from the marine aquaculture tail water discharge ditch of a South American white shrimp breeding farm in Dinghai District, Zhoushan City, Zhejiang Province, and the total nitrogen (TN) content was 10mg / L.

[0094] 3L of water was taken from the marine aquaculture tail water discharge ditch of a South American white shrimp breeding farm in Dinghai District, Zhoushan City, Zhejiang Province, and the total nitrogen (TN) content was 10mg / L. 600 The results showed that the H1R1 strain could tolerate salinity of 15-75g / L. Figure 6 The results showed that the H1R1 strain could tolerate salinity of 15-75g / L.

Claims

1. Pseudomonas taeanensis H1R1, which is preserved in the China General Microbiological Culture Collection Center, has a preservation number of CGMCC No. 34390, a preservation date of April 28, 2025, and a preservation address of No. 3, Yikhina, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Chinese Academy of Sciences.

2. The application of Pseudomonas taeanensis H1R1 in claim 1 in treating mariculture tail water.

3. Use according to claim 2, wherein the compound is ###0002### The application is to reduce the total nitrogen content in mariculture tail water.

4. The use according to claim 3, wherein the compound is ###0002### The method of the application is: (1) inoculate Pseudomonas taeanensis H1R1 into liquid LB enrichment medium, enrich culture at 20-30°C and 100-150 r / min, and obtain bacterial liquid; the liquid LB enrichment medium consists of 10 g / L of tryptone, 5 g / L of yeast extract, 0.1 g / L of KNO3, 35 g / L of NaCl, and deionized water as solvent, and the pH is 7; (2) inoculate the bacterial liquid of step (1) into mariculture tail water, stir at 20-25°C and 130-150 r / min until the total nitrogen removal rate is higher than 80%, and the mariculture tail water is discharged in accordance with the standard.

5. The use according to claim 4, wherein the compound is ###0002### The enrichment culture condition in step (1) is enrichment culture at 25°C and 150 r / min for 48 h.

6. The use according to claim 4, wherein the compound is ###0002### Step (1) OD of bacteria solution 600 was 0.5-1.

3.

7. The use according to claim 4, wherein the compound is ###0002### The bacterial liquid is inoculated into mariculture tail water in an amount of 1-5% by volume concentration.

8. The use according to claim 4, wherein the compound is ###0002### The mariculture tail water contains 10-100 g / L of NaCl, 1-10 mg / L of amino nitrogen, and 10-30 mg / L of nitro nitrogen.

9. The use according to claim 4, wherein the compound is ###00003### 8 or ###00004### 9. The mariculture tail water is simulated mariculture tail water with a final concentration consisting of 0.07215 g / L of KNO3, 0.1688 g / L of C4H6O4, 0.00527 g / L of KH2PO4, 0.00764 g / L of NH4Cl, 0.05 g / L of NaHCO3, and 35.0 g / L of NaCl, and deionized water as solvent.

Images