CN119242540B

CN119242540B - Pseudomonas WH-3 and its application

Info

Publication number: CN119242540B
Application number: CN202411774387.6A
Authority: CN
Inventors: 黄会; 徐英江; 王共明; 张秀珍; 陈建强; 王小涵; 胡乃波; 王明磊; 韩慧宗; 张健; 孙琰晴; 刘永春
Original assignee: Shandong Marine Resource and Environment Research Institute
Current assignee: Shandong Marine Resource and Environment Research Institute
Priority date: 2024-12-05
Filing date: 2024-12-05
Publication date: 2025-05-16
Anticipated expiration: 2044-12-05
Also published as: CN119242540A

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

Pseudomonas WH-3 and application thereof

Technical Field

The invention relates to marine bacteria and application thereof, in particular to pseudomonas WH-3 and application thereof in degradation of metolachlor and 2-methyl-6-ethylaniline (MEA), and belongs to the technical field of microorganisms.

Background

Metolachlor is a chloroacetamide selective herbicide with a molecular formula of C ₁₅H₂₂ClNO₂. The metolachlor has the characteristics of broad spectrum, high efficiency, strong selectivity and the like, and is widely applied to dry crops, vegetable crops, orchards, paddy rice transplanting fields and the like.

2-Methyl-6-ethylaniline (MEA) is an intermediate for synthesizing chloroacetamides such as metolachlor, acetochlor and the like, is also an important metabolite for photodecomposition, hydrolysis and biodegradation of chloroacetamides, can exist and accumulate in a living body for a long time in a natural environment, has strong toxicity, and is harmful to living beings and human health.

The amide herbicide is frequently detected in the domestic marine environment, the detection concentration and the detection rate of the metolachlor in the sea water in the yellow river delta shellfish enrichment culture area are both higher, and moderate risks exist in part of stations. The metolachlor is detected in the downstream river channels such as the small clear river near the eastern nutrient Guangrao and the sea water and sediment in the adjacent sea areas to different degrees, the detection rate in the sea water exceeds 50 percent, and the partial area is as high as 91.4 percent. Alachlor is detected from the sea water near the opal sea area. The metolachlor is detected from the coastal sea shellfish in Shandong, so that the pollution is expanded to the marine ecological environment.

The toxicity of the amide herbicide to aquatic animals is mainly shown as teratogenesis, death, enzyme activity influence and the like, and the toxicity to aquatic plants is mainly shown as reduction of plant cell biomass, interference of cell division, inhibition of photosynthesis and the like, and the potential marine environment ecological risk caused by the large-scale use of the amide herbicide is not ignored. The chemical property of the chloroacetamide herbicide is stable, the herbicide can exist in marine environment and marine organisms for a long time, and the metolachlor and MEA are slowly degraded in the environment, wherein the hydrolysis half-life of the metolachlor in the seawater in summer is 67 days, the non-biodegradation half-life of the metolachlor in summer is 50 days, the hydrolysis half-life of the metolachlor in the seawater in winter is 277 days, and the non-biodegradation half-life of the metolachlor in winter is 193 days. At present, the pollution repair work of herbicides in marine environments is an important point in recent years. Therefore, there is a need to develop a rapid, efficient, safe repair method for chloroacetamide herbicide pollution in marine environments.

Patent application CN116042443a discloses an anaerobic degradation strain (Trichococcus sp. SRB-2) having an anaerobic degradation half-life of 2.0 days for metolachlor at a concentration of 20mg·l ^-1, which is not related to the degradation of MEA by the strain.

The existing literature (separation, identification and degradation characteristic research of the metolachlor degradation strain Y4-6) (Gaoshan university of Nanjing, gaoshan university paper Dong, 2013) discloses a pseudomonas strain (Pseudoxanthomonas sp.) which can degrade the metolachlor and does not relate to the degradation condition of the metolachlor to MEA.

It can be seen that in the existing pesticide degrading microorganisms, no strain capable of degrading metolachlor and MEA simultaneously is seen.

Disclosure of Invention

Aiming at solving the defects of the prior art, the invention aims to provide marine bacteria which can degrade metolachlor and MEA simultaneously and has high degradation efficiency and wide adaptation salinity.

In order to achieve the above object, the present invention adopts the following technical scheme:

pseudomonas WH-3, latin name Pseudomonas juntendi WH-3, is preserved in China general microbiological culture Collection center, with preservation date of 2024, 08 and 02, and preservation number of CGMCC NO.31535, and the place of preservation is Beijing, china.

The application of the pseudomonas WH-3 in degrading metolachlor and 2-methyl-6-ethylaniline.

The invention has the advantages that the Pseudomonas WH-3 provided by the invention is used for separating and screening seawater polluted by the metolachlor in sea area, the strain is wide in adaptation salinity and can degrade the metolachlor and MEA with high efficiency, and has high-efficiency bioremediation effect on marine environment polluted by the metolachlor and MEA (including seawater and marine sediment), wherein after the strain is used for treating the seawater polluted by the metolachlor (with the concentration of 50 mg.L ^-1) and MEA (with the concentration of 50 mg.L ^-1) for 7 days, the biodegradation rates of the metolachlor and MEA respectively reach 88.1 percent, 82.7 percent, the degradation half lives are respectively 2.67 days and 3.53 days, and after the strain is used for treating the marine sediment polluted by the metolachlor (with the concentration of 50 mg.kg ^-1) and MEA (with the concentration of 50 mg.kg ^-1), the biodegradation rates of the metolachlor and MEA respectively reach 82.7 percent, 78.6 percent, and the degradation half lives are respectively 3.27 days and 4.02 days.

Drawings

FIG. 1 is a colony morphology of Pseudomonas WH-3;

FIG. 2 is a diagram showing the shape of the cells of Pseudomonas WH-3;

FIG. 3 is a phylogenetic tree of Pseudomonas WH-3;

FIG. 4 is a graph showing the results of strain growth detection of Pseudomonas WH-3 at various temperatures;

FIG. 5 is a graph showing the results of strain growth measurements of Pseudomonas WH-3 at different salinity;

FIG. 6 is a graph showing the results of strain growth detection of Pseudomonas WH-3 at various pH values;

FIG. 7 is a graph showing the statistical results of the degradation rates of Pseudomonas WH-3 on metolachlor and MEA at different inoculum sizes;

FIG. 8 is a graph showing the statistical results of the biodegradation rates of metolachlor and MEA in a contaminated seawater sample;

FIG. 9 is a graph of the biodegradation rate statistics of metolachlor and MEA in contaminated marine sediment samples.

Detailed Description

The invention is described in detail below with reference to the drawings and the specific embodiments.

1. Sample collection

For 10 months in 2022, a sample of surface seawater was collected from a site (N38 DEG 07'28.86' ', E118 DEG 13'51.07 '') on the offshore area of yellow river delta contaminated with metolachlor and filtered with a sterile filter membrane, the filter membrane was placed into a 100mL sterile centrifuge tube, the cap was screwed, sealed with a sealing membrane, and the tube was transported in an ice box to a laboratory for use.

2. Enrichment, screening, separation and purification of strains

1. Preparation of culture medium

The enrichment medium was prepared by weighing 19.45g of sodium chloride, 5.98g of magnesium chloride, 3.24g of sodium sulfate, 1.80g of calcium chloride, 0.55g of potassium chloride, 0.16g of sodium carbonate, 0.08g of potassium bromide, 0.034g of strontium chloride, 0.022g of boric acid, 0.004g of sodium silicate, 0.0024g of sodium fluoride, 0.0016g of sodium nitrate and 0.008g of disodium hydrogen phosphate, dissolving them in 1000mL of distilled water under heating, and adjusting the pH to 7.6. 100mL of enrichment medium is taken and split into 250mL conical flasks, 20 small glass beads with a diameter of 1mm are added into each flask, and the mixture is autoclaved at 121 ℃ for 20min for later use.

Preparing 1000mL of enrichment medium, adding 15.0g of agar, heating for dissolution, packaging into 250mL conical flasks, autoclaving at 121 ℃ for 20min, and pouring into a plate for later use.

Preparing a medicine-containing flat-plate culture medium, namely preparing 1000mL of enrichment culture medium, adding 100mg of metolachlor, 100mg of MEA and 15.0g of agar, heating and dissolving, subpackaging into 250mL conical flasks, sterilizing at 121 ℃ for 20min, and pouring into a flat plate for later use.

10G of tryptone, 5g of yeast extract and 19.45g of sodium chloride (the concentration of sodium chloride in the conventional LB medium is 5 g.L ^-1～10g·L^-1) are weighed out, dissolved in 1000mL of distilled water, and the pH value is adjusted to 7.0. 100mL of the modified LB culture medium is taken and split into 250mL conical flasks, and the mixture is autoclaved at 121 ℃ for 20min for later use.

2. Enrichment and acclimatization of strains

And simultaneously adding the metolachlor and the MEA into the enrichment medium, and enabling the concentration of the metolachlor and the MEA to be 20 mg.L ^-1. Cutting the obtained filter membrane loaded with microorganisms in the seawater sample, adding the filter membrane into the enrichment medium added with the metolachlor and the MEA, carrying out enrichment culture at 30 ℃ and 150 r.min ^-1, taking the culture every 7 days, transferring the culture into a new enrichment medium at a transferring amount of 5%, and gradually increasing the concentration of the metolachlor and the MEA in the enrichment medium to 40 mg.L ^-1、60mg·L^-1、80mg·L^-1、100mg·L^-1 every transferring. After each transfer, culturing for 24 hours, sampling, sending to a Shandong water product quality inspection center, detecting the residual quantity of metolachlor and MEA through gas chromatography mass spectrometry, and judging the degradation degree of metolachlor and MEA.

Table 1 degradation rates of metolachlor and MEA by microorganisms on the filter membrane

As shown in Table 1, the degradation rates of the culture on the metolachlor and the MEA gradually increased within the same time period along with the increase of the domestication passage times and the concentration of the metolachlor and the MEA, and the degradation rates of the culture on the metolachlor and the MEA with the concentration of 100 mg.L ^-1 in the 5 th generation reach 59.6% and 57.3% respectively.

3. Isolation and purification of strains

After 4 times of continuous domestication and passage of microorganisms on the filter membrane, 1mL of 5 th generation culture is taken for gradient dilution, 100 mu L of dilution liquid with dilution multiple of 10 ^-3、10^-4、10^-5、10^-6 is taken and respectively coated on a medicine-containing flat plate culture medium (respectively marked as a flat plate 1, a flat plate 2, a flat plate 3 and a flat plate 4), and the mixture is placed in a 30 ℃ constant temperature incubator for 7 days for culture.

Compared with other plates, the colonies on the plate 2 are more in variety and moderate in number, a plurality of single colonies with different forms and colors are picked from the plate 2, and are respectively and continuously streaked and cultured for 3 times to obtain purified strains, and the strain with the best growth vigor is selected from the purified strains and is marked as WH-3.

The metolachlor and MEA were added simultaneously to the modified LB medium and the concentrations of metolachlor and MEA were set to 100 mg.L ^-1, and strain WH-3 was inoculated into the modified LB medium with metolachlor and MEA added as described above and cultured on a 150 r.min ^-1 shaker at 30℃for 24 hours, and then was maintained with 30% glycerol (-70℃for preservation) as a backup strain.

3. Colony morphology, cell shape and physiological and biochemical characteristics of strain WH-3

1. Colony morphology

Under aseptic conditions, strain WH-3 was inoculated into a non-medicated plate medium by a three-wire method, and after culturing for 48 hours, as shown in FIG. 1, it was observed that a milky-white circular colony with a convex center was formed on the medium, with a diameter of about 1.5mm, a smooth surface and a blurred edge.

2. Shape of the fungus body

Two loops of freshly cultured strain WH-3 were taken with an inoculating loop, added to 1mL of sterile water, and mixed well. And (3) covering the copper holes special for the two electron microscopes with the bacterial suspension for 3min, drying bacterial liquid by using filter paper, dyeing for 1.5min by using phosphotungstic acid, and drying the phosphotungstic acid by using the filter paper. The shape of the cells was observed under Hitachi H-7650 transmission electron microscope, and as shown in FIG. 2, strain WH-3 was in the form of a short rod without flagella.

3. Physiological and biochemical characteristics

By the identification of an API 20E bacteria identification system, the strain WH-3 can assimilate and utilize arginine, sodium citrate and pyruvate, can oxidize glucose, rhamnose, melibiose and arabinose, cannot assimilate and utilize o-nitrobenzene-galactoside, sodium thiosulfate, kohn gelatin, mannitol, inositol, sorbitol, sucrose and amygdalin, cannot hydrolyze lysine, ornithine and tryptophan and cannot produce indole.

4. Species identification of strain WH-3

Extracting genome DNA by adopting a proteinase K cleavage method, and taking 3 mu L for electrophoresis detection. The Marker is DL9000, the bands are 9000bp, 5000bp, 3000bp, 2000bp, 1000bp and 500bp in sequence from top to bottom, the loading amount is 3 mu L, the bright band is 30ng mu L ^-1, the rest bands are 10ng mu L ^-1, and P1, P2 and P3 are extraction reagent blank controls.

A16S rDNA fragment of strain WH-3 was amplified using 16S full-length amplification forward primer 8F (nucleotide sequence: 5'-AGAGTTTGATCCTGGCTCAG-3') and reverse primer 8R (nucleotide sequence: 5 '-TACGGYTACCTTGTTAYGACTT-3'), and the amplified product was detected by 1% agarose gel electrophoresis and sent to the Feifen Standard technical service Co., ltd for sequencing.

The sequencing results were subjected to homology analysis using a BLAST search system and the 16S rDNA sequences of related species collected in the GenBank nucleic acid database, and a phylogenetic tree of the strain WH-3 was constructed using the MEGA 7.0 software using the neighbor-joining method, and the construction results are shown in FIG. 3.

By comparison, strain WH-3 was a Pseudomonas strain clustered with Pseudomonas juntendi and forming independent internal branches, and therefore, strain WH-3 was designated Pseudomonas juntendi WH-3.

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

1. Growth detection of strain WH-3 at different temperatures

Mu.L of WH-3 bacterial liquid cultured to an exponential phase is inoculated into a modified LB culture medium, and the culture is carried out on a 150 r.min ^-1 shaking table at 10 ℃, 15 ℃,20 ℃, 25 ℃,30 ℃, 35 ℃, 40 ℃ and 45 ℃ for 24 hours respectively, 3 parallel groups are arranged in each experiment, the OD ₆₀₀ of the culture medium at the initial stage and the OD ₆₀₀ at the 24 th stage of the experiment are measured by using an enzyme-labeling instrument, and the strain growth amount is expressed as a difference (delta OD ₆₀₀) between the OD ₆₀₀ of the culture medium at the 24 th stage and the OD ₆₀₀ of the culture medium at the initial stage of the experiment.

The results of strain growth measurements of strain WH-3 at different temperatures are shown in FIG. 4. As shown in FIG. 4, the strain WH-3 can grow at 10 ℃ to 45 ℃ and the optimal growth temperature is 30 ℃.

2. Growth detection of strain WH-3 under different salinity

The amounts of sodium chloride added to the modified LB medium were adjusted to 0g, 10g, 20g, 30g, 40g, 50g, 60g, 70g, 80g, 90g and 100g (dissolved in 1000mL of distilled water), respectively, so that the salinity of the medium was 0, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% and 10%, respectively, 500. Mu.L of WH-3 bacterial liquid cultured to the exponential phase was inoculated into the medium having different salinity, and the culture was carried out on a 150 r.min ^-1 shaker at 30℃for 24 hours, 3 replicates were set for each experiment, and the OD ₆₀₀ of the medium at the initial stage and 24h of the experiment were measured by using an enzyme-labeled instrument, and the strain growth was expressed as the difference (. DELTA.OD ₆₀₀) between the OD ₆₀₀ of the medium at the 24h and the OD ₆₀₀ of the medium at the initial stage of the experiment.

The results of the growth measurements of strain WH-3 at different salinity are shown in FIG. 5. As shown in FIG. 5, the salinity range of the strain WH-3 is 0-10%, and the optimal salinity is 3% -4%.

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

The pH values of the improved LB culture media were respectively adjusted to 3,4, 5, 6,7, 8, 9 and 10, 500 mu L of WH-3 bacterial liquid cultured to an exponential phase was inoculated into the culture media with different pH values, and the culture media were cultured on a 150 r.min ^-1 shaking table at 30 ℃ for 24 hours, 3 parallel groups were set for each experiment, OD ₆₀₀ of the culture media at the initial time of the experiment and at the 24 th time were measured by using an enzyme-labeling instrument, and the strain growth amount was expressed as the difference (. DELTA.OD ₆₀₀) between OD ₆₀₀ of the culture media at the 24 th time and OD ₆₀₀ of the culture media at the initial time of the experiment.

The results of the growth measurements of strain WH-3 at different pH values are shown in FIG. 6. As shown in FIG. 6, the pH value of the strain WH-3 is 3-10, and the optimal growth pH value is 7-8.

In summary, the strain WH-3 can grow in the conditions of 10 ℃ to 45 ℃ C, pH of 3-10 and 0-10% of sodium chloride concentration (w/v), the optimal growth temperature is 30 ℃, the optimal growth pH value is 7-8, the optimal salinity is 3% -4%, and the strain WH-3 has the characteristics of wide temperature range, strong salt and acid and alkali resistance and the like, and is suitable for being used in different sea environments.

6. Degradation effect of strain WH-3 on metolachlor and MEA in marine environment

The improved LB culture medium is optimized and regulated according to the optimal salinity (3% -4%) of the strain WH-3, specifically, the dosage of sodium chloride is further increased from 19.45g to 30g, namely, 10g of tryptone, 5g of yeast extract and 30g of sodium chloride are weighed and dissolved in 1000mL of distilled water, and the pH value is regulated to 7.0, so that the high-salt LB culture medium is obtained. And (3) subpackaging 100mL of high-salt LB culture medium into 250mL conical flasks, and autoclaving at 121 ℃ for 20min for later use.

1. Degradation rate of metolachlor and MEA by strain WH-3 under different inoculum sizes

(1) Degradation rate of strain WH-3 on metolachlor under different inoculation amounts

Adding metolachlor into a sterilized high-salt LB culture medium, enabling the concentration of the metolachlor to be 50 mg.L ^-1, inoculating bacterial strains WH-3 into the high-salt LB culture medium added with the metolachlor according to 0.5%, 1%, 3%, 5%, 10%, 15% and 20% of inoculum size, culturing the same for 24 hours on a 150 r.min ^-1 shaking table at 30 ℃, sampling the same for 24 hours, and sending the same to a Shandong water quality inspection center to detect the residual quantity of the metolachlor through gas chromatography mass spectrum, and judging the degradation degree of the metolachlor.

(2) Degradation rate of MEA by strain WH-2 at different inoculum sizes

MEA was added to the sterilized high-salt LB medium and the MEA concentration was set to 50 mg.L ^-1, and strain WH-3 was inoculated to the above-mentioned high-salt LB medium supplemented with MEA in an inoculum size of 0, 0.5%, 1%, 3%, 5%, 10%, 15%, 20%, 3 in each group, and incubated at 30℃for 24 hours on a 150 r.min ^-1 shaker, and samples were taken at 24 hours and sent to the Shandong provincial aquatic products quality inspection center to detect the residual amount of MEA by gas chromatography mass spectrometry, to determine the degradation degree of MEA.

The results of the biodegradation rate statistics of the strain WH-3 on metolachlor and MEA under different inoculation amounts are shown in FIG. 7. As can be seen from FIG. 7, the degradation efficiency of both metolachlor and MEA was significantly improved with the increase of the inoculation amount of the strain WH-3, and the degradation efficiency was balanced when the inoculation amount exceeded 5%, so that the optimal inoculation amount of the strain WH-3 was 5%.

2. Culture of strain WH-3

The strain WH-3 is inoculated into a high-salt LB culture medium and cultured for 24 hours at 30 ℃ on a 150 r.min ^-1 shaking table to obtain a fermentation broth.

3. Preparation of contaminated seawater samples and marine sediment samples

And simultaneously adding the metolachlor and the MEA into the seawater to ensure that the concentration of the metolachlor and the MEA is 50 mg.L ^-1, thereby obtaining a polluted seawater sample.

The method comprises the steps of simultaneously adding metolachlor and MEA to ocean sediment, and enabling the concentration of the metolachlor and the MEA to be 50 mg.kg ^-1, so that a polluted ocean sediment sample is obtained.

4. Treatment of contaminated samples with strain WH-3

The fermentation broth of strain WH-3 was added to the contaminated seawater sample and the contaminated marine sediment sample, respectively, at an inoculum size of 5% (v/v), and left to stand at 30℃for 7 days.

During the standing treatment, the concentrations of metolachlor and MEA were sampled every 24 hours, and the biodegradation rate and degradation half-life of metolachlor and MEA were calculated.

5. Results

The statistical results of the biodegradation rates of metolachlor and MEA in the contaminated seawater samples are shown in FIG. 8. As can be seen from FIG. 8, after the polluted seawater sample is treated by the strain WH-3 for 7 days, the biodegradation rate of metolachlor is 88.1%, the degradation half-life is 2.67 days, the biodegradation rate of MEA is 82.7%, and the degradation half-life is 3.53 days.

The statistics of the biodegradation rate of metolachlor and MEA in the contaminated marine sediment samples are shown in fig. 9. As can be seen from FIG. 9, after the contaminated marine sediment sample was treated with the strain WH-3 for 7 days, the biodegradation rate of metolachlor was 82.7%, 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.

The results show that the strain WH-3 has high-efficiency bioremediation effect on marine environments (including seawater and marine sediments) polluted by metolachlor and MEA.

7. Safety of strain WH-3 on marine animals and marine microalgae

The strain WH-3 is inoculated into a high-salt LB culture medium, and is subjected to shaking culture at 30 ℃ and 150 r.min ^-1 to reach an exponential phase, and is centrifuged at 5000 r.min ^-1 for 5min, and thalli are collected into a sterile PBS buffer solution for later use.

1. Safety of strain WH-3 on marine shellfish

Mactra veneriformis (Mactra veneriformis) comes from the eastern coast of Shandong, is temporarily cultured in natural seawater (water temperature 25+/-0.5 ℃) for 7 days, is fed with the small crescent diamond algae 2 times per day and water is changed 2 times per day during temporary culture, is continuously inflated for 2/3 of each time, is used for timely cleaning feces, picking out dead and unhealthy individuals, and is stopped feeding 1 day before an experiment, and the uniform and healthy shellfish are selected as experimental objects, and are divided into an experimental group and a control group, wherein 3 parallel shellfish are arranged in each group, and 30 shellfish are arranged in each group.

Strain WH-3 is added into the water body of the experimental group to a final concentration of 10 ⁷CFU·mL^-1, the same dose of sterile PBS buffer solution is added into the water body of the control group, the Nicotiana microcystis is fed for 1 time every 24 hours during the experiment, water is changed for 1 time, water is changed for 2/3 time each time, aeration is continuously carried out, feces are cleaned in time, and continuous observation is carried out for 7 days.

The observation result shows that all the shellfish groups are healthy and active and have no death.

2. Safety of strain WH-3 on ocean copepods

Both the Chinese salt-water fleas (Halicyclops SINENSIS KIEFER) and the Pacific spindle fleas (ACARTIA PACIFICA) were harvested from the yellow river delta offshore area. Natural seawater is filtered by a 0.45 mu m mixed cellulose filter membrane and used, the water temperature is 25+/-0.5 ℃, and the light-dark ratio is L:D=12 h:12 h. After 3 days of domestication culture, 300 healthy and active adults are selected for experiments, an experiment group and a control group are arranged, each group is provided with 3 parallels, and 50 parallels are all filled in a 250mL conical flask.

Strain WH-3 is added into the water body of the experimental group to a final concentration of 10 ⁷CFU·mL^-1, the same dose of sterile PBS buffer solution is added into the water body of the control group, rhodotorula marina is fed for 1 time every 24 hours during the experiment period, and the water body is subjected to micro-aeration and continuous observation for 3 days.

As a result, each group of daphnia survived normally without death.

3. Safety of strain WH-3 on marine microalgae

The Chlorella (Nitzschia closterium f. Minissima) is provided by the sea resource and environmental institute algae room in Shandong province. The natural seawater and the f/2 culture solution are sterilized and then used for culturing the Nicotiana microcephala, the water temperature is 25+/-0.5 ℃, the illumination intensity is 3000lx, and the light-dark ratio is L:D=12 h:12 h. Pre-culturing for 3 generations, and performing experiments after microscopic examination of normal cells and entering a logarithmic growth phase. An experimental group and a control group are arranged, each group is provided with 3 parallels, and 100mL of logarithmic growth phase algae liquid is taken from each parallels and filled into a 250mL conical flask.

Strain WH-3 was added to the experimental group water to a final concentration of 10 ⁷CFU·mL^-1, the same dose of sterile PBS buffer was added to the control group water, and the continuous observation was performed for 3 days, and the number of algal cells was counted under a microscope using a hemocytometer.

The statistical result shows that the number of the algae cells in each group has no obvious difference.

The results show that strain WH-3 is relatively safe for marine shellfish, marine copepods and marine microalgae at a concentration of 10 ⁷CFU·mL^-1 and below.

8. Preservation of bacterial species

Pseudomonas WH-3, latin name Pseudomonas juntendi WH-3, is sent to China general microbiological culture Collection center (CGMCC) for preservation, the preservation date is 2024, 08 and 02 days, the preservation number is CGMCC NO.31535, and the place of preservation is Beijing, china.

It should be noted that the above examples are only examples for clearly illustrating the present invention, and are not limiting to the embodiments of the present invention. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. Not all embodiments are exhaustive. Obvious changes and modifications which are extended by the technical proposal of the invention are still within the protection scope of the invention.

Claims

1. Pseudomonas WH-3, Latin name Pseudomonas juntendi WH-3, deposited in the General Microbiology Center of China Microorganism 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.