Pseudomonas putida, microbial agent thereof and application of pseudomonas putida in chemical wastewater treatment

Abstract

The invention discloses Pseudomonas putida, a bacterial agent thereof and an application of the Pseudomonas putida in chemical wastewater treatment, the Pseudomonas putida is named as PH-20 and preserved in the China General Microbiological Culture Collection Center on March 17, 2025, and the preservation number of the strain is CGMCC No. 33831. The invention further discloses a preparation method of the Pseudomonas putida. The pseudomonas putida disclosed by the invention can be used for rapidly degrading polybenzoic acid such as terephthalic acid, phthalic acid, isophthalic acid, trimellitic acid, trimellitic acid and pyromellitic acid in wastewater under the conditions of high salt and low temperature, and the degradation effect is still excellent under the condition that various pollutants coexist.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to a Pseudomonas putida, a bacterial agent thereof, and an application thereof in the treatment of chemical industrial wastewater. Background Art

[0002] In the field of chemical production, polybasic benzoic acid pollutants, as important chemical intermediates, are widely used in multiple industries such as polyimide, medicine, and plasticizers. However, chemical industrial wastewater often contains such substances and a large amount of salts. The discharged untreated wastewater will cause harm to the environment and the human body. Although the current biochemical treatment technology is recognized as the most economical and effective means for treating water environmental pollution, the existing methods for obtaining microbial bacterial agents (including directional purification culture, enrichment screening, and directional culture by activated sludge method) have obvious limitations. Although the purified cultured strains have high purity, high concentration, and fast culture speed, they are easily poisoned by the high-salt and highly toxic environment when put into actual chemical industrial wastewater, resulting in poor adaptability or even death of the strains. Moreover, the low-temperature environment (such as below 15 °C) will significantly inhibit the activity of specific domesticated strains. Although the operating temperature can be improved by heat preservation and heating, the inhibitory effect of environmental factors on the strains cannot be completely eliminated. In view of this, it is necessary to study a strain that can tolerate high-salt conditions and low-temperature conditions and rapidly degrade polybasic benzoic acid in chemical industrial wastewater. Summary of the Invention

[0003] To solve the deficiencies of the prior art, the purpose of the present invention is to provide a Pseudomonas putida that can effectively degrade terephthalic acid, phthalic acid, isophthalic acid, trimellitic acid, trimesic acid, and pyromellitic acid in wastewater under high-salt conditions and low-temperature conditions.

[0004] To achieve the above object, the present invention adopts the following technical scheme: A Pseudomonas putida, the Pseudomonas putida ( Pseudomonas putida ) is named PH-20 and was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on March 17, 2025, and the deposit number of the strain is: CGMCC No. 33831.

[0005] The application of Pseudomonas putida in degrading polybasic benzoic acid in chemical industrial wastewater, the polybasic benzoic acid is one or more of terephthalic acid, phthalic acid, isophthalic acid, trimellitic acid, trimesic acid, and pyromellitic acid, the concentration of polybasic benzoic acid in the chemical industrial wastewater is 0.5 - 5 g / L, the NaCl concentration is 5 - 30 g / L, and the temperature is 8 - 35 °C.

[0006] A bacterial agent produced by using the above-mentioned Pseudomonas putida.

[0007] Application of the above microbial agent in degrading polybasic benzoic acid in chemical industrial wastewater, wherein the polybasic benzoic acid is one or more of terephthalic acid, phthalic acid, isophthalic acid, trimellitic acid, mesitylenic acid and pyromellitic acid, the concentration of polybasic benzoic acid in the chemical industrial wastewater is 0.5 - 5 g / L, the NaCl concentration is 5 - 30 g / L, and the temperature is 8 - 35 °C.

[0008] A method for treating chemical industrial wastewater with a microbial agent, wherein a microbial agent with a volume ratio of 3% - 7% is added to the chemical industrial wastewater for degradation treatment.

[0009] A preparation method of a microbial agent, comprising the following specific steps: S1. Inoculate Pseudomonas putida PH - 20 into an LB medium and shake - culture until the logarithmic phase; S2. Inoculate the cultured strain into a seed tank and culture until the logarithmic growth phase to obtain a seed solution; S3. Connect the seed solution to a production tank for fermentation culture to obtain a microbial agent.

[0010] Preferably, in step S1, the formula of the LB medium is: NaCl 10.00 g / L, peptone 10.00 g / L, yeast powder 5.00 g / L, pyromellitic acid 1 g / L, pH 7.0.

[0011] Preferably, in step S2, the formula of the medium used in the seed tank is: pyromellitic acid 1 g / L, glucose 8 g / L, (NH 4 ) 2 SO 4 1 g / L, K 2 HPO 4 2 g / L, MgSO 4 0.5 g / L, NaCl 1 g / L, CaCO 3 0.5 g / L, yeast extract 2 g / L, pH 7.2 - 7.5.

[0012] Preferably, in step S3, the medium used in the production tank is the same as that in the seed tank; during the culture processes of steps S2 and S3, the inoculation amount is 8% - 12%, the ventilation volume of sterile air is 1:0.6 - 1.2, the stirring speed is 180 - 240 r / min, the culture temperature is 30 - 35 °C, and the culture time is 48 - 60 h.

[0013] The beneficial effects of the present invention are as follows: The Pseudomonas putida of the present invention can effectively degrade polybasic benzoic acids such as terephthalic acid, phthalic acid, isophthalic acid, trimellitic acid, mesitylenic acid and pyromellitic acid in wastewater under high - salt conditions and low - temperature conditions, and still has excellent degradation effects in wastewater with multiple co - existing polybasic benzoic acid pollutants and high - concentration polybasic benzoic acid pollutants, and has a wide application range. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 is a colony morphology diagram of Pseudomonas putida bacteria in the present invention; Figure 2 This is the degradation rate of polybenzoic acid pollutants by Pseudomonas putida; Figure 3 The effect of NaCl concentration on the degradation efficiency of strain PH-20; Figure 4 The effect of pH on the degradation efficiency of strain PH-20; Figure 5 is the effect of temperature on the degradation efficiency of strain PH-20; Figure 6 The effect of the initial concentration of pyromellitic acid on the degradation efficiency of strain PH-20; Figure 7 This is a diagram showing the effect of strain PH-20 in treating actual chemical wastewater. DETAILED DESCRIPTION

[0015] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0016] Example 1, Isolation and identification of strains: Take 3.0 mL of activated sludge obtained from the wastewater treatment pool of a chemical enterprise (one of the main products of the enterprise is pyromellitic acid) and add it to 100 mL of inorganic salt medium containing pyromellitic acid (pyromellitic acid concentration is 200 mg / L), the formula of which is: NaCl 5.0 g, (NH 4 ) 2 SO 4 1.0 g, K 2 HPO 4 •7H 2 O 1.96 g, KH 2 PO 4 0.5 g MgSO 4 •7H 2 O0.2 g, make up to 1000 mL with deionized water, pH 7.0-7.2, sterilize at 121 ℃ for 20 min, culture with shaking at 10 ℃, transfer to fresh inorganic salt culture medium at a 3% inoculum every 10 days, and transfer continuously for 5 times.

[0017] Take 1.0 mL of the enriched bacterial solution obtained above and add 9.0 mL of sterile water to make 10 -1 Then draw 1.0 mL of the 10 -1 Add the enriched solution to 9.0 mL of sterile water and mix thoroughly to make 10 -2Enrichment solution, and so on, and the enrichment solution is serially diluted. Pipette 0.1 mL of each dilution and spread it on an inorganic salt solid medium containing 200 mg / L of pyromellitic acid (formulation as above), and culture at 30 °C for 10 days. After 10 days, pick single colonies from the above inorganic salt solid medium, culture in 3.0 mL of LB liquid medium for 24 hours. The formula of the LB liquid medium is: pyromellitic acid 500 mg / L, NaCl 10.00 g / L, peptone 10.00 g / L, yeast powder 5.00 g / L, pH 7.0; centrifuge at 8000 r / min for 2 min, pour off the supernatant, add 3.0 mL of sterile water and shake well, still centrifuge at 8000 r / min for 2 min, wash twice with sterile water according to this method, and then add 3.0 mL of sterile water to resuspend the bacteria. Pipette 1.0 mL of this bacterial solution and add it to 100 mL of an inorganic salt liquid medium containing 200 mg / L of pyromellitic acid (formulation: containing 200 mg of pyromellitic acid per liter, 1.50 g of K 2 HPO 4 、0.50 g of KH 2 PO 4 、0.20 g of MgSO 4 •7H 2 O、1.00 g of NaCl、1.00 g of (NH 4 ) 2 SO 4 、20.00 g of agar, pH 7.0), culture by shaking at 160 r / min and 30 °C for 96 h, and measure its degradation effect by gas chromatography. One strain with higher degradation efficiency was preserved, namely Pseudomonas putida PH-20 (deposit number: CGMCC No. 33831), and its colony morphology on the LB solid medium is as Figure 1 shown, and subsequent experiments were carried out.

[0018] The main physiological characteristics of Pseudomonas putida are: the cell shape is rod-shaped, arranged singly or in pairs, with a size of about (0.5~0.8) μm × (1.5~3.0) μm, having a terminal flagellum, strong motility, and negative Gram staining. The 16S rRNA gene sequence of Pseudomonas putida is shown in SEQ ID No. 1.

[0019] The above strain was deposited in the China General Microbiological Culture Collection Center on March 17, 2025. Its taxonomic name is Pseudomonas putida ( Pseudomonas putida ), the strain name is PH-20, the deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the deposit number of the strain is: CGMCC No. 33831.

[0020] Example 2. A preparation method of a bacterial agent, comprising the following specific steps: S1. Inoculate Pseudomonas putida PH-20 into LB medium. The formula of the LB medium is: 10.00 g / L of NaCl, 10.00 g / L of peptone, 5.00 g / L of yeast powder, 1 g / L of pyromellitic acid, pH 7.0, and shake culture until the logarithmic phase; S2. Inoculate the cultured bacterial strain into a seed tank. The formula of the medium used in the seed tank is: 1 g / L of pyromellitic acid, 8 g / L of glucose, (NH 4 ) 2 SO 4 1 g / L, K 2 HPO 4 2 g / L, MgSO 4 0.5 g / L, 1 g / L of NaCl, CaCO 3 0.5 g / L, 2 g / L of yeast extract, pH 7.2 - 7.5, and culture until the logarithmic growth phase, namely the seed liquid; S3. Inoculate the seed liquid into a production tank for fermentation culture. The medium used in the production tank is the same as that in the seed tank to obtain the bacterial agent.

[0021] Among them, during the culture processes of steps S2 and S3, the inoculation amount is 8% - 12%, the ventilation amount of sterile air is 1:0.6 - 1.2, the stirring speed is 180 - 240 r / min, the culture temperature is 30 - 35 °C, and the culture time is 48 - 60 h.

[0022] Example 3. Degradation effect of strain PH-20 on polybenzoic acid pollutants: Prepare inorganic salt liquid media containing terephthalic acid, phthalic acid, isophthalic acid, trimellitic acid, hemimellitic acid, and pyromellitic acid (the initial concentration is 500 mg / L for all), and the formula of the medium is: (NH 4 ) 2 SO 4 1.0 g, K 2 HPO 4 •7H 2 O 1.96 g, KH 2 PO 4 0.5 g, MgSO 4 •7H 20.2 g of O, with a NaCl concentration of 10 g / L, pH 5.0, autoclaved at 121 °C for 20 minutes, and left to stand until room temperature. The PH-20 seed solution was inoculated into the culture media of each pollutant at an inoculation amount of 5% (v / v), and cultured with shaking at 35 °C and 180 r / min for 96 h, and the content of each pollutant was measured, as Figure 2 shown.

[0023] As can be seen from Figure 2 , the degradation efficiency of strain PH-20 for terephthalic acid, phthalic acid, isophthalic acid, trimellitic acid, pyromellitic acid and mellitic acid is all above 95%, and the degradation effect is excellent.

[0024] Example 4. Effect of NaCl concentration on the degradation efficiency of strain PH-20: The NaCl concentration in the inorganic salt medium was adjusted to 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L and 30 g / L respectively. Taking pyromellitic acid as the model pollutant, 500 mg / L of pyromellitic acid was added, (NH 4 ) 2 SO 4 1.0 g, K 2 HPO 4 •7H 2 O 1.96 g, KH 2 PO 4 0.5 g, MgSO 4 •7H 2 O 0.2 g, pH 5.0, autoclaved at 121 °C for 20 minutes, and left to stand until room temperature. The above seed solution was inoculated into a serum bottle at an inoculation amount of 5%, and cultured on a shaker at 30 °C and 180 r / min for 96 h, and then the different degradation efficiencies of pyromellitic acid in the solution were monitored. The results are shown in Figure 3 .

[0025] As can be seen from Figure 3 , strain PH-20 has good tolerance to salt concentration, and can achieve efficient degradation of pyromellitic acid within the tested NaCl concentration range. When the NaCl concentration is 30 g / L, the degradation efficiency can still exceed 80% within 96 h, which indicates that strain PH-20 has good tolerance to high-salt conditions and is suitable for the treatment process of high-salt chemical wastewater.

[0026] Example 5. Effect of pH on the degradation efficiency of strain PH-20: In the culture medium containing 500 mg / L of pyromellitic acid, the components of the culture medium are: (NH 4 ) 2 SO 4 1.0 g, K2 HPO 4 •7H 2 O 1.96 g, KH 2 PO 4 0.5 g, MgSO 4 •7H 2 O 0.2 g. Autoclave at 121 °C for 20 minutes and let it stand until room temperature. Add the PH-20 seed solution to the culture media of each pollutant at an inoculation amount of 5% (v / v). Adjust the pH values to 4.0, 5.0, 6.0, 7.0, and 8.0 respectively with 1 mol / L hydrochloric acid and sodium hydroxide. After shaking culture at 30 °C and 180 rpm for 96 h, measure the pollutant content as Figure 4 shown.

[0027] As Figure 4 can be seen, in the range of pH 4.0 - 8.0, the degradation rate of prehnitic acid by strain PH-20 is above 85%, indicating that the strain has strong applicability to acid and alkali.

[0028] Example 6. Effect of temperature on the degradation efficiency of strain PH-20: Prepare a culture medium containing 500 mg / L prehnitic acid. The components of the culture medium are: (NH 4 ) 2 SO 4 1.0 g, K 2 HPO 4 •7H 2 O 1.96 g, KH 2 PO 4 0.5 g, MgSO 4 •7H 2 O 0.2 g, pH 5.0. Autoclave at 121 °C for 20 minutes and let it stand until room temperature. Add the PH-20 seed solution to the culture media of each pollutant at an inoculation amount of 5% (v / v). Place them at 5 °C, 8 °C, 15 °C, 25 °C, and 35 °C respectively, and shake culture at 180 rpm for 96 h. Then measure the pollutant content as Figure 5 shown.

[0029] As Figure 5 can be seen, in the range of 5 - 35 °C, the degradation rate of prehnitic acid by strain PH-20 increases with the increase of temperature. When the degradation temperature is 8 - 15 °C, the degradation rate can still reach more than 70%. The results show that strain PH-20 has good low-temperature tolerance.

[0030] Example 7. Effect of the initial concentration of prehnitic acid on the degradation efficiency of strain PH-20: Prepare media with pyromellitic acid concentrations of 0.5, 1, 1.5, 2, 2.5, and 5 g / L respectively. The components of the medium are: (NH 4 ) 2 SO 4 1.0 g, K 2 HPO 4 •7H 2 O 1.96 g, KH 2 PO 4 0.5 g, MgSO 4 •7H 2 O 0.2 g, pH 5.0. Autoclave at 121 °C for 20 minutes and let it stand until room temperature. Add the PH-20 seed liquid to the media of each pollutant at an inoculation amount of 5% (v / v) respectively, and place them in a shaker at 30 °C and 180 rpm for 96 h, then measure the pollutant content, as Figure 6 shown.

[0031] As Figure 6 can be seen, the degradation rate of the strain PH-20 for pyromellitic acid decreases with the increase of the initial concentration of pyromellitic acid. When the concentration is 5 g / L, the degradation rate can reach 80.6%. This shows that the strain PH-20 also has excellent degradation effect in the wastewater containing high-concentration polybenzoic acid pollutants and has a wide application range.

[0032] Example 8. Degradation application of strain PH-20 in actual chemical wastewater: The water source for treatment is the wastewater treatment pool of a chemical enterprise, with a treatment volume of 10 L. Add the bacterial agent prepared from the strain PH-20 to the sewage at a dosing amount of 5% by volume. The original sewage has a pH of about 6.0 and a temperature of 25 °C. The initial concentrations of trimellitic acid, trimesic acid, pyromellitic acid, and COD in the sewage are 1600 mg / L, 1500 mg / L, 1200 mg / L, and 5000 mg / L respectively. Aerate the sewage to make the dissolved oxygen above 2 mg / L, and detect the concentrations of each pollutant in the wastewater every 12 h, as Figure 7 shown.

[0033] As Figure 7 can be seen, after adding the strain PH-20 for 72 h, the degradation rates of trimellitic acid, trimesic acid, pyromellitic acid, and COD in the wastewater all reach over 90%. The above experimental data show that the strain PH-20 has excellent degradation effect on polybenzoic acid pollutants in actual chemical wastewater, and the degradation rate of the strain PH-20 is still very high in the case of coexistence of multiple pollutants, and it has good application prospects in actual chemical wastewater.

[0034] The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and all technical solutions obtained by means of equivalent replacement or equivalent transformation fall within the protection scope of the present invention.

Claims

1. A strain of Pseudomonas putida, characterized in that The Pseudomonas putida ( Pseudomonas putida ) was named PH-20 and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on March 17, 2025. The deposit number of the strain is: CGMCC No. 33831.

2. Use of the Pseudomonas putida according to claim 1 in degrading polybenzoic acid in chemical wastewater, characterized in that: The polybenzoic acid is one or more of terephthalic acid, phthalic acid, isophthalic acid, trimellitic acid, trimesic acid and pyromellitic acid. The concentration of polybenzoic acid in chemical wastewater is 0.5-5 g / L, the concentration of NaCl is 5-30 g / L, and the temperature is 8-35 °C.

3. A bacterial agent produced using the Pseudomonas putida described in claim 1.

4. Use of the bacterial agent according to claim 3 in degrading polybenzoic acid in chemical wastewater, characterized in that: The polybenzoic acid is one or more of terephthalic acid, phthalic acid, isophthalic acid, trimellitic acid, trimesic acid and pyromellitic acid. The concentration of polybenzoic acid in chemical wastewater is 0.5-5 g / L, the concentration of NaCl is 5-30 g / L, and the temperature is 8-35 °C.

5. A method for treating chemical wastewater using the bacterial agent according to claim 3, characterized in that: Add 3%~7% of bacterial agent by volume into chemical wastewater for degradation treatment.

6. A method for preparing the bacterial agent according to claim 3, characterized in that: The specific steps include: S1. Inoculate Pseudomonas putida PH-20 into LB medium and culture with shaking until the logarithmic phase; S2, inoculating the cultured bacteria into a seed tank and culturing them to the logarithmic growth phase, i.e., the seed solution; S3. Connect the seed liquid to the production tank for fermentation and culture to obtain the bacterial agent.

7. The method for preparing the bacterial agent according to claim 6, characterized in that: In the step S1, the formula of the LB medium is: NaCl 10.00 g / L, peptone 10.00 g / L, yeast powder 5.00 g / L, pyromellitic acid 1 g / L, pH 7.

0.

8. The method for preparing the bacterial agent according to claim 6, characterized in that: In the step S2, the culture medium formula used in the seed tank is: 1 g / L of pyromellitic acid, 8 g / L of glucose, 1 g / L of (NH4)2SO4, 2 g / L of K2HPO4, 0.5 g / L of MgSO4, 1 g / L of NaCl, 0.5 g / L of CaCO3, 2 g / L of yeast extract, and pH 7.2-7.

5.

9. The method for preparing the bacterial agent according to claim 8, characterized in that: In the step S3, the culture medium used in the production tank has the same formula as the culture medium in the seed tank; during the culture process of steps S2 and S3, the inoculation amount is 8%~12%, the ventilation volume of sterile air is 1:0.6~1.2, the stirring speed is 180~240 r / min, the culture temperature is 30~35°C, and the culture time is 48~60 h.

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