Pseudomonas WH-3 and its application

Abstract

The present invention discloses Pseudomonas WH‑3 and its application, belonging to the field of microbial technology. The Pseudomonas WH‑3, Latin name Pseudomonas juntendi WH‑3, is deposited in the General Microbiological Center of China Microbiological Culture Collection Administration Committee, with a deposit date of August 2, 2024, a deposit number of CGMCC No.31535, and a deposit location in Beijing, China. The Pseudomonas WH‑3 provided by the present invention is separated and screened from seawater contaminated by metolachlor. The strain is adapted to a wide range of salinities, can efficiently degrade metolachlor and 2‑methyl‑6‑ethylaniline, and has an efficient bioremediation effect on marine environments polluted by metolachlor and 2‑methyl‑6‑ethylaniline.

Description

Technical Field

[0001] The invention relates to a marine bacterium and an application thereof, in particular to a Pseudomonas WH-3 and an application thereof in degrading metolachlor and 2-methyl-6-ethylaniline (MEA), belonging to the technical field of microorganisms. Background Art

[0002] Isopropylamine is a chloroacetamide selective herbicide with a molecular formula of C 15 H 22 ClNO2. Because of its broad spectrum, high efficiency and strong selectivity, isopropylamine is widely used in dryland crops, vegetable crops, orchards and rice transplanting fields.

[0003] 2-Methyl-6-ethylaniline (MEA) is an intermediate in the synthesis of chloroacetamides such as isopropylamine and acetochlor. It is also an important metabolite of the photolysis, hydrolysis and biodegradation of chloroacetamides. It can exist and accumulate in organisms for a long time in the natural environment, and is highly toxic, causing harm to organisms and human health.

[0004] Amide herbicides are frequently detected in the domestic marine environment. The concentration and detection rate of isopropylamine in the seawater of the shellfish aquaculture area in the Yellow River Delta are both high, and some stations are at moderate risk. Isopropylamine has been detected in seawater and sediments in the downstream rivers such as Xiaoqing River near Guangrao, Dongying, and its adjacent sea areas to varying degrees, and the detection rate in seawater exceeds 50%, and in some areas it is as high as 91.4%. Isopropylamine has been detected in seawater near the Rushan sea area. Isopropylamine has been detected in marine shellfish along the Shandong coast, indicating that its pollution has spread to the marine ecological environment.

[0005] The toxicity of amide herbicides to aquatic animals is mainly manifested in teratogenicity, lethality, and effects on enzyme activity, and the toxicity to aquatic plants is mainly manifested in reducing plant cell biomass, interfering with cell division, inhibiting photosynthesis, etc. The potential marine environmental ecological risks caused by the large-scale use of amide herbicides cannot be ignored. Chloroacetamide herbicides are chemically stable and can exist in the marine environment and marine organisms for a long time. Isopropylamine and MEA degrade slowly in the environment. Among them, the hydrolysis half-life of isopropylamine in summer seawater is 67 days and the non-biodegradation half-life is 50 days, and the hydrolysis half-life in winter seawater is 277 days and the non-biodegradation half-life is 193 days. At present, the pollution remediation of herbicides in the marine environment has been the focus of attention in recent years. Therefore, it is urgent to develop a fast, efficient and safe method for remediating chloroacetamide herbicide pollution in the marine environment.

[0006] Patent application CN116042443A discloses an anaerobic degradation strain ( Trichococcus sp. SRB-2), the strain was sensitive to the concentration of 20 mg·L -1The anaerobic degradation half-life of isopropylamine was 2.0 days, and the degradation of MEA by this strain was not involved.

[0007] The existing literature "Isolation, identification and degradation characteristics of isopropylamine-degrading strain Y4-6" (Master's degree thesis of Nanjing Agricultural University, Dong Yang, 2013) discloses a strain of Pseudomonas ( Pseudoxanthomonas sp. ), this strain can degrade isopropylamine, and the degradation of MEA by this strain is not involved.

[0008] It can be seen that among the existing pesticide-degrading microorganisms, there is no strain that can degrade isopropylamine and MEA at the same time. Summary of the invention

[0009] In order to solve the deficiencies of the prior art, the object of the present invention is to provide a marine bacterium which can simultaneously degrade isopropylamine and MEA and has high degradation efficiency and can adapt to a wide range of salinities.

[0010] In order to achieve the above object, the present invention adopts the following technical solution:

[0011] Pseudomonas WH-3, whose Latin name is Pseudomonas juntendi WH-3, is deposited in the General Microbiology Center of China Culture Collection Administration. The deposit date is August 2, 2024. The deposit number is CGMCCNO.31535. The deposit unit is Beijing, China.

[0012] The aforementioned Pseudomonas WH-3 is used for the degradation of metolachlor and 2-methyl-6-ethylaniline.

[0013] The present invention is beneficial in that: the Pseudomonas WH-3 provided by the present invention is separated and screened from seawater polluted by isopropylamine, the strain is adaptable to a wide range of salinities, can efficiently degrade isopropylamine and MEA, and has a highly efficient bioremediation effect on the marine environment (including seawater and marine sediments) polluted by isopropylamine and MEA. The strain is used to treat the marine environment polluted by isopropylamine (concentration of 50 mg·L -1 ) and MEA (concentration of 50 mg·L -1 ) after 7 days, the biodegradation rates of metolachlor and MEA reached 88.1% and 82.7%, respectively, and the degradation half-lives were 2.67 days and 3.53 days, respectively. -1 ) and MEA (concentration of 50 mg kg -1 ) After 7 days in the polluted marine sediments, the biodegradation rates of isopropylamine and MEA reached 82.7% and 78.6%, respectively, and the degradation half-lives were 3.27 days and 4.02 days, respectively. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a picture of the colony morphology of Pseudomonas WH-3;

[0015] Figure 2 This is a diagram of the bacterial shape of Pseudomonas WH-3;

[0016] Figure 3 is the phylogenetic tree diagram of Pseudomonas WH-3;

[0017] Figure 4 This is a graph showing the growth test results of Pseudomonas WH-3 at different temperatures;

[0018] Figure 5 This is a graph showing the growth test results of Pseudomonas WH-3 strains at different salinities;

[0019] Figure 6 This is a graph showing the growth test results of Pseudomonas WH-3 strain at different pH values;

[0020] Figure 7 It is the statistical result diagram of the degradation rate of metolachlor and MEA by Pseudomonas WH-3 at different inoculation amounts;

[0021] Figure 8 It is a statistical result graph of biodegradation rate of isopropylamine and MEA in contaminated seawater samples;

[0022] Fig. 9 It is a statistical graph of the biodegradation rates of isopropylamine and MEA in contaminated marine sediment samples. DETAILED DESCRIPTION

[0023] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0024] 1. Sample collection

[0025] In October 2022, surface seawater samples were collected from a station in the nearshore waters of the Yellow River Delta contaminated with isopropylamine (N38°07′28.86″, E118°13′51.07″) and filtered with a sterile filter membrane. The filter membrane was placed in a 100mL sterile centrifuge tube, the lid was tightened and sealed with a sealing film, placed in an ice box and transported to the laboratory for use.

[0026] 2. Strain enrichment, screening, isolation and purification

[0027] 1. Culture medium preparation

[0028] Enrichment medium: weigh 19.45g sodium chloride, 5.98g magnesium chloride, 3.24g sodium sulfate, 1.80g calcium chloride, 0.55g potassium chloride, 0.16g sodium carbonate, 0.08g potassium bromide, 0.034g strontium chloride, 0.022g boric acid, 0.004g sodium silicate, 0.0024g sodium fluoride, 0.0016g sodium nitrate and 0.008g disodium hydrogen phosphate, heat and dissolve in 1000mL distilled water, and adjust the pH value to 7.6. Take 100mL of enrichment medium and dispense it into 250mL conical flasks, add 20 small glass beads with a diameter of 1mm to each flask, sterilize it by high pressure at 121℃ for 20min, and set aside.

[0029] Drug-free plate culture medium: prepare 1000 mL of enriched culture medium, add 15.0 g of agar, heat to dissolve and dispense into 250 mL conical flasks, sterilize at 121°C for 20 min, pour into plates and set aside.

[0030] Drug-containing plate culture medium: prepare 1000 mL of enrichment culture medium, add 100 mg of isopropylamine, 100 mg of MEA and 15.0 g of agar, heat to dissolve and dispense into 250 mL conical flasks, sterilize at 121°C for 20 min, pour into plates and set aside.

[0031] Improved LB medium: weigh 10 g tryptone, 5 g yeast extract and 19.45 g sodium chloride (the sodium chloride concentration of conventional LB medium is 5 g L -1 ~10g·L -1 ), dissolved in 1000 mL of distilled water, and the pH value was adjusted to 7.0. Take 100 mL of the modified LB medium and dispense it into a 250 mL conical flask, sterilize it by high pressure at 121°C for 20 min, and set aside.

[0032] 2. Enrichment and domestication of strains

[0033] Metolachlor and MEA were added to the enrichment medium at the same time, and the concentrations of both metolachlor and MEA were 20 mg·L -1 The filter membrane containing microorganisms in the seawater sample obtained above was cut into pieces and added to the enrichment medium supplemented with isopropylamine and MEA. -1 Enrichment culture was performed. Every 7 days, the culture was transferred to a new enrichment medium at a transfer rate of 5%. Each time the concentration of isopropylamine and MEA in the enrichment medium was gradually increased to 40 mg·L -1 、60mg·L -1 、80mg·L -1 、100mg·L -1After each transfer, samples were taken after 24 hours of incubation and sent to Shandong Provincial Aquatic Product Quality Inspection Center for gas chromatography-mass spectrometry to detect the residual amounts of isopropylamine and MEA and determine the degradation degree of isopropylamine and MEA.

[0034] Table 1 Degradation rates of metolachlor and MEA by microorganisms on the filter membrane

[0035]

[0036] As shown in Table 1, with the increase of the number of acclimation passages and the concentrations of isopropylamine and MEA, the degradation of isopropylamine and MEA by the culture gradually increased within the same period of time. The degradation of isopropylamine and MEA by the 5th generation culture was 100 mg·L -1 The degradation rates of isopropylamine and MEA reached 59.6% and 57.3% respectively.

[0037] 3. Isolation and purification of strains

[0038] After the microorganisms on the filter membrane were continuously domesticated and subcultured for 4 times, 1 mL of the 5th generation culture was taken for gradient dilution. 100 μL of 10 -3 , 10 -4 , 10 -5 , 10 -6 The diluted solutions of different dilution multiples were spread on drug-containing plate culture media (respectively marked as plate 1, plate 2, plate 3, and plate 4), and cultured in a constant temperature incubator at 30°C for 7 days.

[0039] Compared with other plates, plate 2 had more types of colonies and a moderate number. Multiple single colonies with different morphologies and colors were picked from plate 2 and streaked and cultured three times to obtain purified strains. The strain with the best growth was selected and recorded as WH-3.

[0040] Metolachlor and MEA were added to the modified LB medium at the same time, and the concentrations of both metolachlor and MEA were 100 mg·L -1 The strain WH-3 was inoculated into the modified LB medium supplemented with isopropylamine and MEA and then grown at 150 r·min -1 The strain was cultured at 30°C on a shaker for 24 h and then preserved in 30% glycerol (-70°C) as a backup strain.

[0041] 3. Colony morphology, bacterial shape, and physiological and biochemical characteristics of strain WH-3

[0042] 1. Colony morphology

[0043] Under sterile conditions, strain WH-3 was inoculated into drug-free plate medium using the three-line method. After 48 h of culture, Figure 1As shown, milky white, circular colonies with a protrusion in the middle, about 1.5 mm in diameter, smooth surface and fuzzy edges were observed to form on the culture medium.

[0044] 2. Bacteria shape

[0045] Take two loops of freshly cultured strain WH-3 with an inoculation loop, add to 1 mL of sterile water, and mix. Take two copper holes for electron microscopy, cover with aspirated bacterial suspension for 3 minutes, dry the bacterial solution with filter paper, stain with phosphotungstic acid for 1.5 minutes, and dry the phosphotungstic acid with filter paper. Observe the shape of the bacteria under a Hitachi H-7650 transmission electron microscope, such as Figure 2 As shown, strain WH-3 is short rod-shaped and has no flagella.

[0046] 3. Physiological and biochemical characteristics

[0047] Identification by the API 20E bacterial identification system showed that strain WH-3 could assimilate and utilize arginine, sodium citrate, and pyruvate, and could oxidize glucose, rhamnose, mesidic acid, and arabinose, but could not assimilate and utilize o-nitrophenyl-galactoside, sodium thiosulfate, Kohn gelatin, mannitol, inositol, sorbitol, sucrose, and amygdalin, and could not hydrolyze lysine, ornithine, and tryptophan, and could not produce indole.

[0048] Species Identification of Strain WH-3

[0049] The genomic DNA was extracted by proteinase K cleavage, and 3 μL was taken for electrophoresis detection. The marker was DL9000, and the bands from top to bottom were 9000bp, 5000bp, 3000bp, 2000bp, 1000bp, and 500bp, respectively. The sample volume was 3 μL, and the bright band was 30 ng·μL -1 , the remaining bands were all 10 ng·μL -1 , P1, P2, and P3 are blank controls of extraction reagents.

[0050] The 16S rDNA fragment of strain WH-3 was amplified using the 16S full-length amplification forward primer 8F (nucleotide sequence: 5'-AGAGTTTGATCCTGGCTCAG-3') and the reverse primer 8R (nucleotide sequence: 5'-TACGGYTACCTTGTTAYGACTT-3'). The amplified product was detected by 1% agarose gel electrophoresis and then sent to Feifan Standard Technology Service Co., Ltd. for sequencing.

[0051] The sequencing results were analyzed for homology using the BLAST search system and the 16S rDNA sequences of related species collected in the GenBank nucleic acid database, and the phylogenetic tree of strain WH-3 was constructed using the MEGA 7.0 software and the Neighbour-joining method. The construction results are shown in Figure 3 shown.

[0052] Comparative analysis showed that strain WH-3 was a Pseudomonas strain, which clustered with Pseudomonas juntendi and formed an independent internal branch. Therefore, strain WH-3 was named Pseudomonas juntendi WH-3.

[0053] 5. Growth of strain WH-3 under different temperature, salinity and pH conditions

[0054] 1. Detection of the growth of strain WH-3 at different temperatures

[0055] Take 500 μL of WH-3 cultured to the exponential phase and inoculate it into the modified LB medium. -1 The cells were cultured on a shaker at 10, 15, 20, 25, 30, 35, 40, and 45 °C for 24 h. Three parallels were set up for each experimental group. The OD of the culture medium was measured at the beginning of the experiment and at 24 h using an ELISA reader. 600 The growth rate of the strain was measured by the OD value of the culture medium at 24 h. 600 The OD of the culture medium at the beginning of the experiment 600 The difference (△OD 600 )express.

[0056] The results of strain growth test of strain WH-3 at different temperatures are as follows Figure 4 As shown. Figure 4 It can be seen that strain WH-3 can grow at 10℃~45℃, and the optimal growth temperature is 30℃.

[0057] 2. Growth detection of strain WH-3 under different salinities

[0058] The amount of sodium chloride added to the modified LB medium was adjusted. Specifically, the amount of sodium chloride added was 0 g, 10 g, 20 g, 30 g, 40 g, 50 g, 60 g, 70 g, 80 g, 90 g, and 100 g (dissolved in 1000 mL of distilled water), respectively, so that the salinity of the culture medium was 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%, respectively. 500 μL of WH-3 bacterial solution cultured to the exponential phase was inoculated into the culture medium with different salinities, and the culture medium was incubated at 150 r·min -1 The culture was incubated at 30°C for 24 h on a shaker. Three parallels were set up for each experimental group. The OD of the culture medium was measured at the beginning of the experiment and at 24 h using an ELISA reader. 600 The growth rate of the strain was measured by the OD value of the culture medium at 24 h. 600 The OD of the culture medium at the beginning of the experiment 600 The difference (△OD 600 )express.

[0059] The growth test results of strain WH-3 under different salinities are as follows Figure 5 As shown. Figure 5 It can be seen that the salinity range for the growth of strain WH-3 is 0-10%, and the optimal salinity is 3%-4%.

[0060] 3. Growth detection of strain WH-3 at different pH values

[0061] The pH value of the modified LB medium was adjusted to 3, 4, 5, 6, 7, 8, 9, and 10, respectively. 500 μL of WH-3 culture solution cultured to the exponential phase was inoculated into the culture medium with different pH values, and the culture medium was incubated at 150 r·min. -1 The culture was incubated at 30°C for 24 h on a shaker. Three parallels were set up for each experimental group. The OD of the culture medium was measured at the beginning of the experiment and at 24 h using an ELISA reader. 600 The growth rate of the strain was measured by the OD value of the culture medium at 24 h. 600 The OD of the culture medium at the beginning of the experiment 600 The difference (△OD 600 )express.

[0062] The growth test results of strain WH-3 at different pH values ​​are as follows Figure 6 As shown. Figure 6 It can be seen that the pH range for the growth of strain WH-3 is 3-10, and the optimal pH value for growth is 7-8.

[0063] In summary, strain WH-3 can grow at 10℃~45℃, pH 3-10, and sodium chloride concentration (w / v) 0-10%. The optimal growth temperature is 30℃, the optimal growth pH is 7-8, and the optimal salinity is 3%-4%. It has the characteristics of a wide temperature range, strong salt resistance and acid and alkali resistance, and is suitable for use in different marine environments.

[0064] VI. Degradation effect of strain WH-3 on metolachlor and MEA in marine environment

[0065] The above-mentioned improved LB medium was optimized and adjusted according to the optimal salinity (3% to 4%) of strain WH-3. Specifically, the amount of sodium chloride was further increased from 19.45g to 30g, that is, 10g of tryptone, 5g of yeast extract and 30g of sodium chloride were weighed and dissolved in 1000mL of distilled water, and the pH value was adjusted to 7.0 to obtain a high-salt LB medium. 100mL of high-salt LB medium was dispensed into a 250mL conical flask, autoclaved at 121℃ for 20min, and set aside.

[0066] 1. Degradation rate of metolachlor and MEA by strain WH-3 at different inoculation rates

[0067] (1) Degradation rate of isopropylamine by strain WH-3 at different inoculation rates

[0068] Add metolachlor to the sterilized high-salt LB medium to a concentration of 50 mg / L -1 The strain WH-3 was inoculated into the high-salt LB medium supplemented with isopropylamine at an inoculum of 0, 0.5%, 1%, 3%, 5%, 10%, 15%, and 20%, respectively. Three replicates were set up in each group and the culture was carried out at 150 r·min -1 The mixture was cultured on a shaker at 30°C for 24 h. Samples were taken at 24 h and sent to Shandong Provincial Aquatic Product Quality Inspection Center for gas chromatography-mass spectrometry detection of the residual amount of metolachlor to determine the degree of degradation of metolachlor.

[0069] (2) Degradation rate of MEA by strain WH-2 at different inoculation rates

[0070] Add MEA to the sterilized high-salt LB medium to a concentration of 50 mg / L -1 The strain WH-3 was inoculated into the high-salt LB medium supplemented with MEA at an inoculum of 0, 0.5%, 1%, 3%, 5%, 10%, 15%, and 20%, respectively. Each group had three parallels and was incubated at 150 r·min -1 The mixture was cultured on a shaker at 30°C for 24 h. Samples were taken at 24 h and sent to Shandong Aquatic Products Quality Inspection Center for gas chromatography-mass spectrometry to detect the residual amount of MEA and determine the degree of MEA degradation.

[0071] The statistical results of the biodegradation rates of metolachlor and MEA by strain WH-3 at different inoculation rates are shown in Figure 7 .Depend on Figure 7 It can be seen that with the increase of the inoculation amount of strain WH-3, the degradation efficiency of isopropylamine and MEA was significantly improved. When the inoculation amount exceeded 5%, the degradation efficiency tended to balance. Therefore, the optimal inoculation amount of strain WH-3 was 5%.

[0072] 2. Cultivation of strain WH-3

[0073] The strain WH-3 was inoculated into high-salt LB medium and heated at 150 r·min -1 The culture was carried out on a shaker at 30°C for 24 h to obtain a fermentation broth.

[0074] 3. Preparation of contaminated seawater samples and marine sediment samples

[0075] Metolachlor and MEA were added to the seawater at the same time, and the concentrations of both metolachlor and MEA were 50 mg·L -1 , and obtain contaminated seawater samples.

[0076] Metolachlor and MEA were added to the marine sediments at the same time, and the concentration of both metolachlor and MEA was 50 mg kg -1 , to obtain samples of contaminated marine sediments.

[0077] 4. Treating contaminated samples with strain WH-3

[0078] The fermentation broth of strain WH-3 was added to contaminated seawater samples and contaminated marine sediment samples, respectively, with an inoculation amount of 5% (v / v), and the samples were left to stand at 30°C for 7 days.

[0079] During the static treatment period, samples were taken every 24 hours to detect the concentrations of isopropylamine and MEA, and the biodegradation rates and degradation half-lives of isopropylamine and MEA were calculated.

[0080] 5. Results

[0081] The statistical results of the biodegradation rates of metolachlor and MEA in contaminated seawater samples are shown in Figure 8 .Depend on Figure 8 It can be seen that after the contaminated seawater samples were treated with strain WH-3 for 7 days, the biodegradation rate of isopropylamine was 88.1%, and the degradation half-life was 2.67 days; the biodegradation rate of MEA was 82.7%, and the degradation half-life was 3.53 days.

[0082] The statistical results of the biodegradation rates of metolachlor and MEA in contaminated marine sediment samples are shown in Fig. 9 .Depend on Fig. 9 It can be seen that after the contaminated marine sediment samples were treated with strain WH-3 for 7 days, the biodegradation rate of isopropylamine was 82.7%, and the degradation half-life was 3.27 days; the biodegradation rate of MEA was 78.6%, and the degradation half-life was 4.02 days.

[0083] The above results indicate that strain WH-3 has a highly efficient bioremediation effect on the marine environment (including seawater and marine sediments) contaminated by isopropylamine and MEA.

[0084] VII. Safety of strain WH-3 to marine animals and marine microalgae

[0085] The strain WH-3 was inoculated into high-salt LB medium and incubated at 30°C and 150 r·min -1 The culture was shaken to the exponential phase at 5000 r / min. -1 Centrifuge for 5 minutes and collect the bacteria into sterile PBS buffer for later use.

[0086] 1. Safety of strain WH-3 to marine shellfish

[0087] Four-horned clams (Mactra veneriformis) were collected from the coast of Dongying, Shandong Province. They were temporarily cultured in natural seawater (water temperature 25±0.5℃) for 7 days. During the period of culture, they were fed with Closterium twice a day and the water was changed twice, with 2 / 3 of the water changed each time. The water was aerated continuously, feces was cleaned in time, and dead and unhealthy individuals were picked out. Feeding was stopped one day before the experiment. Healthy clams of uniform size were selected as experimental subjects and divided into experimental and control groups. Each group had 3 parallels, and each parallel had 30 clams.

[0088] The strain WH-3 was added to the water of the experimental group to a final concentration of 10 7 CFU·mL -1 The same dose of sterile PBS buffer was added to the control group water. During the experiment, the fish were fed with Nitzschia closterium once every 24 hours and the water was changed once, with 2 / 3 of the water changed each time. The water was continuously aerated and the feces were cleaned in time. The experiment was conducted for 7 consecutive days.

[0089] Observation results: All the shellfish in each group were healthy and vigorous, with no mortality.

[0090] 2. Safety of strain WH-3 to marine copepods

[0091] Both Halicyclops sinensis Kiefer and Acartiapacifica were collected from the coastal waters of the Yellow River Delta. Natural seawater was filtered through a 0.45 μm mixed cellulose filter membrane and used. The water temperature was 25 ± 0.5 °C and the light-dark ratio was L: D = 12 h: 12 h. After acclimation and culture for 3 days, 300 healthy and active adults were selected for the experiment. The experimental group and the control group were set up, with 3 parallels in each group and 50 in each parallel, all of which were placed in 250 mL conical flasks.

[0092] The strain WH-3 was added to the water of the experimental group to a final concentration of 10 7 CFU·mL -1 The same dose of sterile PBS buffer was added to the control group water. Marine red yeast was fed once every 24 hours during the experiment, and the water was slightly aerated and observed for 3 consecutive days.

[0093] Observation results: Daphnia in each group survived normally and no death was observed.

[0094] 3. Safety of strain WH-3 to marine microalgae

[0095] Nitzschia closterium f.minutissima was provided by the Algae Seed Room of Shandong Institute of Marine Resources and Environment. Natural seawater and f / 2 culture medium were sterilized and used for the culture of Nitzschia closterium f.minutissima. The water temperature was 25±0.5℃, the light intensity was 3000lx, and the light-dark ratio was L∶D =12h∶12h. After three generations of pre-culture, the cells were normal under microscopic examination and the experiment was carried out in the logarithmic growth phase. Experimental and control groups were set up, with 3 parallels in each group. 100mL of logarithmic growth phase algae solution was taken from each parallel and placed in a 250mL conical flask.

[0096] The strain WH-3 was added to the water of the experimental group to a final concentration of 10 7 CFU·mL -1 The same dose of sterile PBS buffer was added to the control group water and observed for 3 consecutive days. The number of algal cells was counted under a microscope using a hemocytometer.

[0097] Statistical results: There was no significant difference in the number of algal cells among the groups.

[0098] The above results show that strain WH-3 7 CFU·mL -1 Concentrations at and below are relatively safe for marine shellfish, marine copepods and marine microalgae.

[0099] 8. Strain Preservation

[0100] Pseudomonas WH-3, whose Latin name is Pseudomonas juntendi WH-3, has been sent to the General Microbiology Center of China Microorganism Culture Collection Administration (CGMCC) for preservation. The preservation date is August 2, 2024, the preservation number is CGMCC NO.31535, and the preservation unit is located in Beijing, China.

[0101] It should be noted that the above embodiments are merely examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the embodiments here. Any obvious changes or modifications derived from the technical solution of the present invention are still within the scope of protection of the present invention.

Claims

1. Pseudomonas WH-3, Latin name Pseudomonas juntendi WH-3 is deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, the deposit date is August 2, 2024, the deposit number is CGMCC No.31535, and the depository address is Beijing, China.

2. Use of the Pseudomonas WH-3 described in claim 1 in the degradation of isopropylamine and 2-methyl-6-ethylaniline.

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