Pseudomonas stutzeri, fungicide and application of pseudomonas stutzeri in degradation of organic nitrogen pollutants in wastewater

Abstract

The invention discloses a pseudomonas stutzeri strain, a microbial agent and application of the pseudomonas stutzeri strain in degradation of organic nitrogen pollutants in wastewater, and belongs to the technical field of microorganisms, the pseudomonas stutzeri strain is named as MDD 2025, and the preservation number of the strain is CGMCC NO. 33839. The pseudomonas stutzeri MDD 2025 can rapidly degrade nitrobenzene, 2, 4-dinitrotoluene, pyridine, aniline and other organic nitrogen pollutants in wastewater under the medium-temperature condition (the water temperature is 35 DEG C) by taking a target pollutant as a unique carbon and nitrogen source, and keeps certain degradation capacity under the low-temperature condition (the water temperature is 10 DEG C).

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microbial technology, and relates to a kind of organic nitrogen pollutant degrading bacteria, in particular to a kind of strain pseudomonas stutzeri, bacterial agent and its application in degrading organic nitrogen pollutants in wastewater. BACKGROUND

[0002] Nitro and amino aromatic compounds are important raw materials and intermediates indispensable in dye, explosive, pesticide and pharmaceutical synthesis, mainly including nitrobenzene, dinitrotoluene, aniline and pyridine, etc. However, due to their stable chemical properties and strong biological toxicity, it is difficult to remove them by conventional physical and chemical methods after entering industrial wastewater, and the residual concentration can be from dozens to hundreds of mg / L, which constitutes a typical characteristic pollutant. As high-nitrogen and high-toxicity organic matter, this kind of substance is not only difficult to be degraded by indigenous microorganisms in the environment, but also can inhibit and poison microbial community, leading to instability and even failure of traditional biological treatment system. Its persistent residue in the natural environment can be amplified through food chain enrichment, and finally endanger ecological safety and human health.

[0003] Currently, the treatment of such wastewater mainly relies on advanced oxidation, adsorption or extraction and other physical and chemical methods. These methods can achieve partial removal, but generally have inherent defects such as high treatment cost, complex process flow, easy production of secondary pollution (such as waste adsorbent and chemical sludge), etc. Traditional biological treatment technology is difficult to achieve stable and efficient purification of such refractory and toxic substances due to the lack of functional microorganisms, low degradation efficiency and weak resistance to impact load of the system. Therefore, it is of urgent and important significance to develop special microbial strain resources that can efficiently and targetedly degrade such high-nitrogen and high-toxicity characteristic pollutants, and to develop biological enhancement technology based thereon, in order to break through the bottleneck of current industrial wastewater treatment. SUMMARY

[0004] The present application provides a kind of strain pseudomonas stutzeri, bacterial agent and its application in degrading organic nitrogen pollutants in wastewater to overcome the defects of prior art.

[0005] To achieve the above purpose, the present application adopts the following technical solutions: In a first aspect, the present application provides a kind of strain pseudomonas stutzeri, which is characterized in that: Pseudomonas stutzeri The strain pseudomonas stutzeri is named MDD 2025, and was deposited at China General Microbiological Culture Collection Center on March 17, 2025, with the strain preservation number of CGMCC NO. 33839.

[0006] The preparation method of liquid enrichment medium for Pseudomonas stearothermia 2025 is as follows: Take 1L of the working solution of the basic medium and autoclave it; after the sterilized working solution of the basic medium is cooled to room temperature, add 10 mL of the filtered and sterilized contaminant mixed stock solution aseptically, and stir thoroughly to obtain the medium.

[0007] The working solution of the basal culture medium has the following formula ( / L): 20 mL phosphate buffer, 10 mL salt and nitrogen source solution, 1 mL trace element solution, and pH adjusted to 7.2 with dilute NaOH or HCl solution. The phosphate buffer has the following formula ( / L): 50 g K₂HPO₄, 50 g KH₂PO₄; the salt and nitrogen source solution has the following formula ( / L): 100 g (NH₄)₂SO₄, 20 g MgSO₄·7H₂O, 5 g NaCl; the trace element solution has the following formula ( / L): 0.5 g FeSO₄·7H₂O, 0.5 g MnSO₄·H₂O, 0.5 g ZnSO₄·7H₂O, 0.25 g CoCl₂·6H₂O, 0.05 g CuSO₄·5H₂O, 0.05 g Na₂MoO₄·2H₂O. The formula of the pollutant mixed mother liquor is as follows ( / L): 1250 mg nitrobenzene, 1250 mg 2,4-dinitrotoluene, 1250 mg pyridine, 1250 mg aniline, and approximately 50-100 mL acetone (chromatographic or analytical grade). The acetone is used to fully dissolve the above solids.

[0008] The preparation method of solid isolation screening medium for Pseudomonas stearothermia 2025 is as follows: add 15 g / L agar powder to the working solution of the basic medium; after autoclaving, place it at 55~60℃, add 10 mL of filtered sterilized contaminant mixed stock solution, shake quickly and gently, and immediately pour the medium into a sterile petri dish to obtain the medium.

[0009] Secondly, the present invention also provides a microbial agent comprising the aforementioned *Pseudomonas schlegelii*.

[0010] Thirdly, the present invention also provides a method for preparing the above-mentioned microbial agent, comprising the following steps: Step 1: Inoculate the *Pseudomonas schlegelii* strain into LB medium and culture with shaking until the logarithmic phase to obtain the strain; Step 2: Inoculate the strain into a seed tank and culture it to the logarithmic growth phase to obtain the seed solution; Step 3: Introduce the seed liquid into the production tank for fermentation and culture to obtain the microbial agent.

[0011] Further, the LB liquid culture medium, the culture medium of the seed tank and the culture medium of the production tank all comprise a contaminant mixed mother liquor; the contaminant mixed mother liquor comprises nitrobenzene, 2,4-dinitrotoluene, pyridine, aniline and acetone.

[0012] Further, the LB liquid culture medium further comprises NaCl, proteose peptone and yeast powder; the culture medium of the seed tank and the culture medium of the production tank are the same and both further comprise glucose, (NH4)2SO4, K2HPO4, MgSO4, NaCl, CaCO3 and yeast paste.

[0013] Further, in the contaminant mixed mother liquor, the amount ratio of nitrobenzene, 2,4-dinitrotoluene, pyridine, aniline and acetone is 1000-1500 mg:1000-1500 mg:1000-1500 mg:1000-1500 mg:50-100 mL, preferably 1250 mg:1250 mg:1250 mg:1250 mg:50-100 mL; the LB liquid culture medium uses water as a solvent, and the concentration of each component is respectively: NaCl 8-12 g / L, proteose peptone 8-12 g / L, yeast powder 3-8 g / L, contaminant mixed mother liquor 8-12 mL / L, preferably NaCl 10.00 g / L, proteose peptone 10.00 g / L, yeast powder 5.00 g / L, contaminant mixed mother liquor 10 mL / L, and the pH is 6.5-7.5, preferably 7.0; the culture medium of the seed tank and the culture medium of the production tank use water as a solvent, and the concentration of each component is respectively: contaminant mixed mother liquor 8-12 mL / L, glucose 6-10 g / L, (NH4)2SO4 0.5-1.5 g / L, K2HPO4 1-3 g / L, MgSO4 0.3-0.8 g / L, NaCl 0.5-1.5 g / L, CaCO3 0.3-0.8 g / L, yeast paste 1-3 g / L, preferably contaminant mixed mother liquor 10 mL, glucose 8 g, (NH4)2SO4 1 g, K2HPO4 2 g, MgSO4 0.5 g, NaCl 1 g, CaCO3 0.5 g, yeast paste 2 g, and the pH is 7.2-7.5.

[0014] Further, in the culture process of the second step and the third step, the inoculation amount is 8-12 %, the ventilation rate of sterile air is 0.6-1.2 vvm, the stirring speed is 180-240 r / min, the culture temperature is 30-35 ℃, and the culture time is 48-60 h.

[0015] In a fourth aspect, the present application further provides the application of the Pseudomonas stutzeri or the bacterial agent described above in degrading organic nitrogen pollutants in wastewater.

[0016] Further, the organic nitrogen type pollutants are nitro or amino aromatic compounds.

[0017] Further, the degradation method is to add the Bacillus aerophilus or the bacterial agent in the chemical industrial wastewater for degradation treatment.

[0018] The application has the advantages that the application provides a Pseudomonas stutzeri, a bacterial agent and application of the Pseudomonas stutzeri in degradation of organic nitrogen type pollutants in wastewater, the Pseudomonas stutzeri MDD 2025 can degrade organic nitrogen type pollutants such as nitrobenzene, 2,4-dinitrotoluene, pyridine and aniline in wastewater at a medium temperature (water temperature 35 DEG C) with the target pollutants as the only carbon and nitrogen source, and can maintain a certain degradation capacity at a low temperature (water temperature 10 DEG C), thereby providing a core strain resource for biological reinforcement technology. The strain can be directly used for treating difficult-to-degrade industrial wastewater such as dyes, explosives and pesticides, can effectively improve the treatment efficiency of a traditional biochemical system, and can guarantee the water quality of effluent. Compared with a physical and chemical method with high cost, the application has the advantages of low treatment cost, environmental friendliness, no secondary pollution and the like, and has significant economic and environmental benefits. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 is a colony morphology diagram of the Pseudomonas stutzeri MDD 2025; Figure 2 is a degradation effect diagram of the Pseudomonas stutzeri MDD 2025 on pyridine, aniline, nitrobenzene and 2,4-dinitrotoluene at a medium temperature and a low temperature. DETAILED DESCRIPTION

[0020] The application is further described below in combination with specific embodiments.

[0021] Embodiment 1 The embodiment provides a Pseudomonas stutzeri, and a separation and identification method is as follows: Take 3.0 mL of activated sludge obtained from a wastewater treatment tank of a certain chemical enterprise in Jiangsu and add the activated sludge into 100 mL of liquid enrichment culture medium. Sterilize at 121 DEG C for 20 min, and culture under oscillation at 10 DEG C, and transfer to fresh inorganic salt culture medium every 10 days at a 3 % inoculation amount, and continuously transfer for 5 times to obtain an enriched bacterial solution.

[0022] Take 1.0 mL of the enriched bacterial solution obtained above, add the enriched bacterial solution into 9.0 mL of sterile water to prepare 10 -1 times enriched solution, and then take 1.0 mL of the prepared 10 -1 times enriched solution and add the enriched solution into 9.0 mL of sterile water to prepare 10 -2The enrichment solution was serially diluted, and so on. 0.1 mL of each dilution was spread onto solid separation and screening medium and incubated at 30 °C for 10 days. After 10 days, a single colony was picked from the solid separation and screening medium and incubated in 3.0 mL of LB liquid medium for 24 hours. Then, it was centrifuged at 8000 r / min for 2 min, the supernatant was discarded, 3.0 mL of sterile water was added and mixed well, and centrifuged again at 8000 r / min for 2 min. After washing twice with sterile water, the bacteria were resuspended in 3.0 mL of sterile water. 1.0 mL of this bacterial suspension and 10 mL of the contaminant mixture were added to inorganic salt liquid medium and incubated at 160 r / min and 30 °C for 96 h with shaking. The degradation effect was then measured by gas chromatography. The strain with the highest degradation efficiency, *Pseudomonas stearothermiae* MDD 2025, was preserved. Its colony morphology on LB solid medium is shown in the figure below. Figure 1 As shown, subsequent experiments were conducted.

[0023] The preparation method for the liquid enrichment medium is as follows: Take 1 L of the basal culture medium working solution and autoclave it; after the sterilized basal culture medium working solution cools to room temperature, aseptically add 10 mL of filtered and sterilized contaminant mixed stock solution, and stir thoroughly to obtain the final product. The formula of the basal culture medium working solution is ( / L): 20 mL phosphate buffer, 10 mL salt and nitrogen source solution, 1 mL trace element solution, and adjust the pH to 7.2 with dilute NaOH or HCl solution. The formulation of phosphate buffer solution is ( / L): 50 g K2HPO4, 50 g KH2PO4; the formulation of salt and nitrogen source solution is ( / L): 100 g (NH4)2SO4, 20 g MgSO4·7H2O, 5 g NaCl; the formulation of trace element solution is ( / L): 0.5 g FeSO4·7H2O, 0.5 g MnSO4·H2O, 0.5 g ZnSO4·7H2O, 0.25 g CoCl2·6H2O, 0.05 g CuSO4·5H2O, 0.05 g Na2MoO4·2H2O. The formula for the mixed mother liquor of pollutants is as follows ( / L): 1250 mg nitrobenzene, 1250 mg 2,4-dinitrotoluene, 1250 mg pyridine, 1250 mg aniline, and approximately 50-100 mL of acetone (chromatographic or analytical grade). Acetone is used to fully dissolve the above solids.

[0024] The preparation method of solid separation screening medium is as follows: add 15 g / L agar powder to the working solution of the basic culture medium; after autoclaving, place it at 55~60℃, add 10 mL of filtered and sterilized contaminant mixed stock solution, shake gently and quickly, and immediately pour the culture medium into a sterile petri dish to obtain the medium.

[0025] The formula of LB liquid medium is ( / L): contaminant mixed stock solution 10 mL, NaCl 10.00 g, protein peptone 10.00 g, yeast powder 5.00 g, and pH is 7.0.

[0026] The formula of inorganic salt liquid medium is ( / L): K2HPO4 1.50 g, KH2PO4 0.50 g, MgSO4·7H2O 0.20 g, NaCl 1.00 g, (NH4)2SO4 1.00 g, and pH is 7.0.

[0027] The formula of LB solid medium is ( / L): contaminant mixed stock solution 10 mL, NaCl 10.00 g, protein peptone 10.00 g, yeast powder 5.00 g, and 20.00 g agar, and pH is 7.0.

[0028] The main physiological characteristics of Pseudomonas stutzeri MDD 2025 are as follows: the cell shape is rod-shaped, arranged singly or in pairs, the size is about (0.6-1.1) μm × (1.8-3.5) μm, with a single polar flagellum, motile, and gram-negative staining. The 16S rRNA gene sequence of Pseudomonas stutzeri MDD 2025 is shown as SEQ ID NO. 1.

[0029] The above strain was deposited with the China General Microbiological Culture Collection Center on March 17, 2025, and was named Pseudomonas stutzeri (Pseudomonas stutzeri) in classification and naming, the strain name is MDD 2025, the deposit address is No. 3, Beichen West Road, Beijing City, Chaoyang District, and the strain preservation number is CGMCC NO. 33839. Pseudomonas stutzeri

[0030] Example 2 The preparation method of the bacterial agent provided in this example comprises the following steps: Step one, inoculate the test tube seed of Pseudomonas stutzeri MDD 2025 into LB liquid medium, the formula of LB liquid medium is ( / L): NaCl 10.00, protein peptone 10.00, yeast powder 5.00, contaminant mixed stock solution 10 mL, and pH is 7.0, and cultivate at 30°C until logarithmic phase to obtain the seed.

[0031] Step two, inoculate the cultivated seed into the seed tank, the formula of the medium used in the seed tank is ( / L): contaminant mixed stock solution 10 mL, glucose 8 g, (NH4)2SO4 1 g, K2HPO4 2 g, MgSO4 0.5 g, NaCl 1 g, CaCO3 0.5 g, and yeast paste 2 g, and pH is 7.2-7.5, and cultivate until logarithmic growth phase to obtain the seed liquid. ​

[0032] Step 3: Introduce the seed liquid into the production tank for fermentation culture. The culture medium used in the production tank is the same as that used in the seed tank to obtain the microbial agent.

[0033] In both steps two and three, the inoculation amount was 8-12%, the sterile air ventilation rate was 0.6-1.2 vvm, the stirring speed was 180-240 r / min, the culture temperature was 30-35 ℃, and the culture time was 48-60 h.

[0034] Example 3 This embodiment provides the application of Pseudomonas stearothermii MDD 2025 in Example 1 and the bacterial agent in Example 2 in the degradation of organic nitrogen pollutants in wastewater.

[0035] The degradation effects of *Pseudomonas stearothermiae* MDD 2025 on nitro and amino aromatic compounds under moderate and low temperatures are as follows: Inorganic salt liquid culture media containing nitrobenzene, 2,4-dinitrotoluene, aniline, and pyridine (initial concentration 5000 mg / L) were prepared separately. The formula of the inorganic salt liquid culture medium ( / L) was: (NH4)2SO4 1.0 g, K2HPO4·7H2O 1.96 g, KH2PO4 0.5 g, MgSO4·7H2O 0.2 g, NaCl concentration 10 g, pH 5.0. The media were autoclaved at 121 ℃ for 20 minutes and allowed to cool to room temperature. MDD 2025 seed culture was added at a 5% inoculum (v / v) to the media containing each contaminant, and cultured at 35 ℃ and 10 ℃ with shaking at 180 r / min for 96 h. The content of each contaminant was measured, and the results are as follows: Figure 2 As shown.

[0036] Depend on Figure 2 It was found that at 35 ℃, *Pseudomonas stearothermiae* MDD 2025 exhibited degradation efficiencies of over 95% for pyridine and aniline, over 90% for p-nitrobenzene, and over 15% for 2,4-dinitrotoluene, demonstrating a significant initiation effect on the degradation of 2,4-dinitrotoluene. At 10 ℃, the degradation rate was generally between 5% and 15% after 96 h, indicating that the strain maintained certain activity at low temperatures.

[0037] In this invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the reagents, materials, and procedures used herein are all widely used in the relevant fields.

[0038] Finally, it should be noted that the above only describes the preferred embodiments of the present application and is not intended to limit the present application. Although the present application has been described in detail with reference to the foregoing embodiments, those skilled in the art will appreciate that the technical solutions described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalent ones. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A strain of *Pseudomonas stearothermii*, characterized by: The *Pseudomonas schlegelii* strain was named MDD 2025 and its preservation number was CGMCC NO. 33839.

2. A microbial agent, characterized in that: It contains *Pseudomonas schrenckii* as described in claim 1.

3. The method for preparing the microbial agent as described in claim 2, characterized in that: Includes the following steps: Step 1: Inoculate the *Pseudomonas schlegelii* strain into LB medium and culture with shaking until the logarithmic phase to obtain the strain; Step 2: Inoculate the strain into a seed tank and culture it to the logarithmic growth phase to obtain the seed solution; Step 3: Introduce the seed liquid into the production tank for fermentation and culture to obtain the microbial agent.

4. The method for preparing the microbial agent according to claim 3, characterized in that: The LB liquid culture medium, the culture medium in the seed tank, and the culture medium in the production tank all contain a mixed mother liquor of contaminants; The pollutant mixture mother liquor includes nitrobenzene, 2,4-dinitrotoluene, pyridine, aniline, and acetone.

5. The method for preparing the microbial agent according to claim 4, characterized in that: The LB liquid culture medium also includes NaCl, peptone, and yeast extract; The culture medium in the seed tank is the same as that in the production tank, both of which also include glucose, (NH4)2SO4, K2HPO4, MgSO4, NaCl, CaCO3 and yeast extract.

6. The method for preparing the microbial agent according to claim 5, characterized in that: In the pollutant mixed mother liquor, the ratio of nitrobenzene, 2,4-dinitrotoluene, pyridine, aniline and acetone is 1000~1500 mg: 1000~1500 mg: 1000~1500 mg: 1000~1500 mg: 50~100 mL. The LB liquid culture medium uses water as a solvent, and the concentrations of each component are as follows: NaCl 8~12 g / L, peptone 8~12 g / L, yeast extract 3~8 g / L, and contaminant mixed stock solution 8~12 mL / L, with a pH of 6.5~7.

5. The culture medium in the seed tank and the culture medium in the production tank use water as a solvent, and the concentrations of each component are as follows: 8-12 mL / L of pollutant mixed mother liquor, 6-10 g / L of glucose, 0.5-1.5 g / L of (NH4)2SO4, 1-3 g / L of K2HPO4, 0.3-0.8 g / L of MgSO4, 0.5-1.5 g / L of NaCl, 0.3-0.8 g / L of CaCO3, and 1-3 g / L of yeast extract, with a pH of 7.2-7.

5.

7. The method for preparing the microbial agent according to claim 3, characterized in that: In the cultivation processes of steps two and three, the inoculation amount is 8-12%, the sterile air ventilation rate is 0.6-1.2 vvm, the stirring speed is 180-240 r / min, the cultivation temperature is 30-35 ℃, and the cultivation time is 48-60 h.

8. The application of *Pseudomonas stearothermii* as described in claim 1 or the bacterial agent as described in claim 2 in the degradation of organic nitrogen pollutants in wastewater.

9. The application according to claim 8, characterized in that: The organic nitrogen pollutants are nitro or amino aromatic compounds.

10. The application according to claim 8, characterized in that: The degradation method is as follows: adding the aerobic Bacillus or the bacterial agent to the chemical wastewater for degradation treatment.

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