Pseudomonas taeanensis H1R1 and application thereof in treating mariculture tail water

Abstract

The application discloses Pseudomonas taishanensis H1R1 and application thereof in treating mariculture tail water, and has excellent high-salt tolerance and aerobic denitrification function, can directly convert ammonia nitrogen and nitrate into nitrogen, efficiently removes nitrogen, and the denitrification rate can reach more than 80%, thereby not only reducing nitrogen load in water, but also helping to prevent water eutrophication caused by nitrogen; can survive and breed under various adverse environmental conditions and treat mariculture tail water. The Pseudomonas taishanensis H1R1 can utilize organic matters in mariculture tail water as carbon source, which not only improves the growth speed and growth amount of the bacteria, but also helps to reduce organic waste in the breeding process, so that the strain H1R1 can promote its own growth while treating mariculture tail water, and realizes double benefits of environmental governance and resource recycling.

Description

(I) Technical Field

[0001] This invention belongs to the field of wastewater treatment technology, specifically involving a new strain of salt-tolerant aerobic denitrifying bacteria—Pseudomonas taianense H1R1 and its application in treating effluent from marine aquaculture. (II) Background Technology

[0002] As a major producer of marine aquaculture, my country's marine aquaculture output has steadily increased year by year, rising from 35.8582 million tons in 2023 to 36.6529 million tons in 2024, an increase of 2.22%. Meanwhile, in the summer of 2023, the area of ​​eutrophic seawater reached 28,960 square kilometers, an increase of 190 square kilometers compared to the previous year. In key marine aquaculture areas, the main pollutant exceeding standards was inorganic nitrogen. The proportions of areas with inorganic nitrogen, reactive phosphate, and chemical oxygen demand exceeding the evaluation standards were 54.2%, 71.8%, and 98.6%, respectively. Therefore, the environmental problems caused by the discharge of marine aquaculture wastewater are increasingly attracting attention. Marine aquaculture wastewater pollutants contain various components such as undigested feed, fish medicine, and metabolic waste from aquaculture organisms. Excessive nitrogen and phosphorus, among other nutrients, can lead to eutrophication and excessive algal blooms. With the increasing emphasis placed on environmental protection by the state, relevant laws and policies have imposed increasingly stringent requirements on the discharge of wastewater from marine aquaculture, necessitating that the marine aquaculture industry strengthen its wastewater treatment practices.

[0003] Patent application CN116062904B discloses a method for treating wastewater and promoting plant growth using a heterotrophic nitrifying-aerobic denitrifying bacterium. The heterotrophic nitrifying-aerobic denitrifying bacterium grown in this method grows rapidly under the following conditions: sodium acetate as carbon source, carbon-nitrogen ratio of 10, pH of 7, culture temperature of 30-40℃, and rotation speed of 180 r / min. However, when treating nitrogen-containing wastewater, the removal efficiency of the strain varies for nitrogen sources of different valence states, and the removal rate of nitrite nitrogen is relatively low, only 18.53%. Patent application CN119372112A discloses a method for treating aerobic denitrifying halophilic bacterium LHT6 in aquaculture wastewater, pharmaceutical wastewater, and other water bodies. The aerobic denitrifying halophilic bacterium LHT6 can grow and remove nitrates under conditions of carbon-to-nitrogen ratio of 2-10, pH of 7-11, and 0%-15% NaCl. It is not only resistant to a variety of antibiotics but can also treat nitrates in water bodies containing various heavy ions. However, the treatment effect of this strain may be affected when treating water bodies with high salinity, such as mariculture tailwater.

[0004] Therefore, it is necessary to screen for a salt-tolerant aerobic denitrifying bacterium to treat marine aquaculture wastewater. (III) Summary of the Invention

[0005] The purpose of this invention is to provide a *Pseudomonas taianense* H1R1 and its application in treating marine aquaculture wastewater. This *Pseudomonas taianense* H1R1 is a salt-tolerant aerobic denitrifying bacterium that can survive and reproduce under various harsh environmental conditions. It can directly convert ammonia nitrogen and nitrate into nitrogen gas, efficiently remove nitrogen, effectively control the total nitrogen content in marine aquaculture wastewater, and solve the problem of high nitrogen content in existing marine aquaculture wastewater.

[0006] The technical solution adopted in this invention is:

[0007] This invention provides a novel salt-tolerant aerobic denitrifying bacterium strain—Pseudomonas staeanensis H1R1, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34390, deposited on April 28, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The *Pseudomonas taianense* H1R1 described in this invention is a salt-tolerant, aerobic, and denitrifying bacterium screened from the bottom mud of a marine aquaculture tailwater discharge ditch in a Litopenaeus vannamei shrimp farm in Dinghai District, Zhoushan City, Zhejiang Province. The colony morphology is nearly circular, white in color, with a raised center, a uniform and moist surface texture, and a clear and regular boundary zone at the edge. Under an electron microscope, the cell is rod-shaped.

[0009] The present invention also provides an application of the aforementioned *Pseudomonas taianense* H1R1 in the treatment of marine aquaculture wastewater, wherein the application is to reduce the total nitrogen content in the marine aquaculture wastewater.

[0010] Further, the application method is as follows: (1) Pseudomonas taianense H1R1 is inoculated into liquid LB enrichment medium and enriched under the conditions of 20-30℃ and 100-150r / min to obtain bacterial solution; the liquid LB enrichment medium is composed of: 10g / L tryptone, 5g / L yeast extract, 0.1g / L KNO3, 35g / L NaCl, water as solvent, pH 7; (2) the bacterial solution in step (1) is inoculated into the effluent of seawater aquaculture and stirred at 20-25℃ and 130-150r / min until the total nitrogen removal rate is higher than 80% and the effluent of seawater aquaculture meets the discharge standards.

[0011] Furthermore, the enrichment culture conditions in step (1) are 25℃ and 150r / min for 48h.

[0012] Further, step (1) bacterial culture OD 600 The value is 0.5-1.3, with 0.8 being preferred.

[0013] Further, in step (2), the bacterial solution is inoculated into the effluent of seawater aquaculture at a volume concentration of 1-5%, preferably 2%.

[0014] Furthermore, the NaCl content in the seawater aquaculture tailwater is 10-100 g / L (preferably 35 g / L), and the amino nitrogen (NH4) content is... + -N) content 1-10 mg / L (preferably 2 mg / L), nitrate nitrogen (NO3) - -N) content 10-30 mg / L (preferably 10 mg / L).

[0015] Furthermore, the seawater aquaculture wastewater is simulated seawater aquaculture wastewater, with the following final concentration composition: 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 invention are mainly reflected in:

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

[0018] (2) The Taian Pseudomonas H1R1 of the present invention can adapt to different salinities, enabling it to survive and reproduce under various harsh environmental conditions and treat marine aquaculture wastewater.

[0019] (3) The Taian Pseudomonas H1R1 of the present invention can use organic matter in the effluent of seawater aquaculture as a carbon source. This not only increases the growth rate and amount of bacteria, but also helps to reduce organic waste in the aquaculture process. This allows strain H1R1 to promote its own growth while treating the effluent of seawater aquaculture, thus achieving the dual benefits of environmental governance and resource recycling. (iv) Description of the attached drawings

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

[0021] Figure 2 Phylogenetic tree of strain H1R1.

[0022] Figure 3 The total nitrogen (TN) and nitrate nitrogen (NO3) of simulated marine aquaculture wastewater were reduced by strain H1R1. - The curve showing the change of N-N content over time.

[0023] Figure 4 The graph shows the growth variation of strain H1R1 and its removal rate of total nitrogen and nitrate nitrogen from simulated marine aquaculture wastewater.

[0024] Figure 5 The curves showing the changes in OD values ​​of simulated marine aquaculture wastewater at different salinities treated with strain H1R1.

[0025] Figure 6 The graph shows the changes in nitrogen removal rate and OD value of actual marine aquaculture tailwater treated by strain H1R1 in Example 4. (V) Detailed Implementation

[0026] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

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

[0028] The culture medium composition in this embodiment of the invention is as follows:

[0029] The liquid BTB medium consisted 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, and a trace element solution of 100 μL / L. The solvent was deionized water, and the pH was 7.0. The trace element solution consisted 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, and CuSO4·5H2O. 1.57 g / L, CoCl2·6H2O 1.61 g / L, solvent is deionized water, pH 6.0.

[0030] Solid BTB medium is liquid BTB medium with 20.0 g / L agar added.

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

[0032] Total nitrogen (TN) was determined by alkaline potassium persulfate digestion-ultraviolet spectrophotometry (refer to the national standard method HJ636-2012).

[0033] Ammonia nitrogen (NH4) + -N) was detected by nanoreagent spectrophotometry (refer to HJ 535-2009).

[0034] Nitrate nitrogen (NO3) - The content of -N was determined by ultraviolet spectrophotometry (refer to HJ / T346-2007).

[0035] Nitrite nitrogen (NO2) - The accumulation of -N was determined by molecular absorption spectrophotometry (refer to GB 7493-87).

[0036] Example 1: Isolation and Identification of Strains H1R1

[0037] 1. Isolation of strain H1R1

[0038] (1) Source of strain

[0039] The strain H1R1 was derived from the bottom mud of the wastewater discharge ditch of a Litopenaeus vannamei shrimp farm in Dinghai District, Zhoushan City, Zhejiang Province.

[0040] (2) Enrichment of strains

[0041] Take 5.0g of sediment and place it in an Erlenmeyer flask containing 50mL of sterile water. After standing at room temperature for a period of time, take 10mL of the supernatant and mix it with 90mL of liquid LB enrichment medium. Incubate for 3 days at a rotation speed of 130r / min and a temperature of 25℃. If the liquid is clear, extend the incubation time; if the liquid is turbid, continue to take 10mL of the supernatant and mix it with 90mL of liquid LB enrichment medium. Repeat the enrichment experiment three times to increase the bacterial concentration.

[0042] (3) Isolation and purification of strains

[0043] The enriched bacterial solution was sequentially rinsed with sterile water for 10 minutes. -1 Up to 10 -9 After gradient dilution, take 1 mL of 10 -4 Up to 10 -6 Gradual dilutions were spread onto solid BTB agar plates and incubated at 25°C for 3 days until colonies formed. Colonies in the blue-colored areas were then selected and isolated by streaking on fresh solid BTB agar plates. This streaking isolation and purification process was repeated three times to obtain purified strains, which were then stored and numbered.

[0044] (4) Screening of strains

[0045] The selected strains were inoculated into new solid LB enrichment medium and incubated statically at 25°C for 48 h. They were then inoculated into liquid BTB medium and incubated with shaking at 25°C and 130 rpm for 72 h, followed by centrifugation. The supernatant was collected and its growth (OD) was measured. 600 ) and NH4 + -N removal rate, NO2 - -N accumulation, NO3 - The removal rates of -N are shown in Table 1. Preliminary selections were made based on good biomass (OD). 600 The strain with the highest nitrogen removal rate (>0.6) is designated as the dominant strain and is denoted 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 onto solid LB enrichment medium and incubated at 25°C for 24 hours. The colonies exhibited a nearly circular outline, a white color, a raised center, a homogeneous and moist surface texture, and a clear, regular boundary at the edge. Under an electron microscope, the cells appeared as rod-shaped (e.g., ...). Figure 1 (As shown).

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

[0053] The purified strain H1R1 was sent to Beijing New Era Zhonghe Technology Co., Ltd. for 16S rDNA sequencing (SEQ ID NO.1). BLAST alignment using the NCBI database showed that the sequence exhibited 100% similarity to both *Pseudomonas sp. strain CS-R1-17* and *Pseudomonas taeanensis* strain JP09. A neighbor-joining phylogenetic tree was constructed using MEGA 11.0. Figure 2 The strain H1R1 was confirmed to belong to the genus Pseudomonas and named Pseudomonas staeanensis H1R1. It was deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34390, on April 28, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.

[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℃ and 130 r / min for 48 h to obtain OD. 600 The bacterial solution has a concentration of 0.8.

[0089] (2) Salt tolerance

[0090] The formula for simulated seawater aquaculture tailwater is the same as in Example 2.

[0091] Five groups of simulated marine aquaculture tailwater were set up with salinity gradients of 15 g / L, 35 g / L, 55 g / L, 75 g / L, and 95 g / L. The bacterial solution prepared in step (1) was added to each group at a volume concentration of 2%. The OD of the bacterial solution was measured after treatment at 25℃ and 130 r / min for 24 h. 600 Values, results are shown below Figure 5 The results showed that strain H1R1 could tolerate salinity of 15–75 g / L.

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

[0093] A 3L sample of water was taken from a ditch used for the discharge of marine aquaculture wastewater from a shrimp farm in Dinghai District, Zhoushan City, Zhejiang Province. The total nitrogen (TN) content was 10mg / L.

[0094] The 3L water body was divided into three parallel groups. The bacterial solution prepared according to the method in Example 3 was added to each water sample at a volume concentration of 2%. The samples were treated at 25℃ and 130 rpm. Samples were taken at 0h, 6h, 12h, 18h, 24h, and 30h to determine the total nitrogen removal rate and the OD of the bacteria in each group. 600 Values, take the average, results are shown in Figure 6 The results showed that the total nitrogen removal rate of strain H1R1 could reach 80%, and its growth was good.

Claims

1. *Pseudomonas taianense* ( Pseudomonas taeanensis H1R1 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34390, on April 28, 2025, at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

2. The application of the *Pseudomonas taianense* H1R1 as described in claim 1 in reducing the amino nitrogen and nitrate nitrogen content in marine aquaculture tailwater.

3. The application as described in claim 2, characterized in that, The application method is as follows: (1) Pseudomonas taianense H1R1 is inoculated into liquid LB enrichment medium and enriched under the conditions of 20-30℃ and 100-150 r / min to obtain bacterial solution; the liquid LB enrichment medium is composed of: 10 g / L tryptone, 5 g / L yeast extract, 0.1 g / L KNO3, 35 g / L NaCl, deionized water as solvent, pH 7; (2) the bacterial solution in step (1) is inoculated into the effluent of seawater aquaculture and stirred at 20-25℃ and 130-150 r / min until the total nitrogen removal rate is higher than 80% and the effluent of seawater aquaculture meets the discharge standards.

4. The application as described in claim 3, characterized in that, The enrichment culture conditions in step (1) are 25℃ and 150 r / min for 48 h.

5. The application as described in claim 3, characterized in that, Step (1) Bacterial OD 600 It ranges from 0.5 to 1.

3.

6. The application as described in claim 3, characterized in that, Step (2) Inoculate the bacterial solution into the tailwater of seawater aquaculture at a volume concentration of 1-5%.

7. The application as described in claim 3, characterized in that, The marine aquaculture tailwater contains 15-75 g / L NaCl, 1-10 mg / L amino nitrogen, and 10-30 mg / L nitrate nitrogen.

8. The application as described in claim 3, characterized in that, The mariculture wastewater was simulated mariculture wastewater, with the following final concentration composition: KNO3 0.07215 g / L, C4H6O4 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.

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