A strain of Pseudomonas putida and its bacterial agent and its application in chemical wastewater treatment

Abstract

The present invention discloses a strain of Pseudomonas putida and a bacterial agent thereof and application thereof in chemical wastewater treatment. Pseudomonas putida ) was named PH-20 and deposited with the General Microbiology Center of the China General Culture Collection Administration on March 17, 2025, with the deposit number: CGMCC No. 33831. The Pseudomonas putida strain of the present invention can rapidly degrade polybenzoic acids such as terephthalic acid, phthalic acid, isophthalic acid, trimellitic acid, pyromellitic acid, and tetracarboxylic acid in wastewater under high-salt and low-temperature conditions, and the degradation effect is still excellent when multiple pollutants coexist.

Description

Technical Field

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

[0002] In the chemical industry, polybenzoic acid pollutants, as important chemical intermediates, are widely used in industries such as polyimide, pharmaceuticals, and plasticizers. However, chemical wastewater often contains these substances and large amounts of salt. Discharge of untreated wastewater poses a threat to both the environment and humans. Although current biochemical treatment technologies are recognized as the most cost-effective means of treating water pollution, existing methods for obtaining microbial agents (including targeted purification, enrichment screening, and activated sludge targeted culture) have significant limitations. While purified strains offer high purity, high concentration, and rapid growth rates, they are susceptible to high salt and toxic environments when introduced into actual chemical wastewater, leading to poor adaptability and even death. Furthermore, low temperatures (e.g., below 15°C) significantly inhibit the activity of specific acclimated strains. While operating temperatures can be improved through insulation and heating, these environmental inhibitory effects cannot be completely eliminated. Therefore, it is necessary to develop a strain that can tolerate high salt and low temperatures and rapidly degrade polybenzoic acids in chemical wastewater. Summary of the Invention

[0003] In order to address the deficiencies of the prior art, the present invention aims to provide a Pseudomonas putida strain that can effectively degrade polybenzoic acids such as terephthalic acid, phthalic acid, isophthalic acid, trimellitic acid, pyromellitic acid and pyromellitic acid in wastewater under high salt and low temperature conditions.

[0004] In order to achieve the above objectives, the present invention adopts the following technical solutions:

[0005] A strain of Pseudomonas putida, Pseudomonas putida ( Pseudomonas putida ) was named PH-20 and deposited in the General Microbiology Center of China Culture Collection Administration on March 17, 2025, with the culture collection number: CGMCC No. 33831.

[0006] The application of Pseudomonas putida in the degradation of polybenzoic acids in chemical wastewater. Polybenzoic acids are one or more of terephthalic acid, phthalic acid, isophthalic acid, trimellitic acid, pyromellitic acid, and tetracarboxylic acid. The concentration of polybenzoic acids in chemical wastewater is 0.5-5 g / L, the NaCl concentration is 5-30 g / L, and the temperature is 8-35 ℃.

[0007] A bacterial agent produced by utilizing the above-mentioned Pseudomonas putida.

[0008] The above-mentioned bacterial agent is used to degrade polybenzoic acid in chemical wastewater, where the polybenzoic acid is one or more of terephthalic acid, phthalic acid, isophthalic acid, trimellitic acid, pyromellitic acid and tetracarboxylic acid. The concentration of polybenzoic acid in the chemical wastewater is 0.5~5 g / L, the NaCl concentration is 5~30 g / L, and the temperature is 8~35°C.

[0009] A method for treating chemical wastewater with a bacterial agent comprises adding a bacterial agent with a volume ratio of 3% to 7% into the chemical wastewater for degradation treatment.

[0010] A method for preparing a microbial agent comprises the following specific steps:

[0011] S1. Inoculate Pseudomonas putida PH-20 into LB medium and culture with shaking until the logarithmic phase;

[0012] S2. Inoculate the cultured bacteria into a seed tank and culture until the logarithmic growth phase, i.e., the seed solution;

[0013] S3. The seed liquid is connected to a production tank for fermentation and culture to obtain a bacterial agent.

[0014] 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.

[0015] Preferably, in 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.

[0016] Preferably, in step S3, the culture medium used in the production tank is the same 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.

[0017] The benefit of the present invention is that the Pseudomonas putida of the present invention can effectively degrade polybenzoic acids such as terephthalic acid, phthalic acid, isophthalic acid, trimellitic acid, 2-methyl-3-pyromellitic acid and 2-methyl-3-pyromellitic acid in wastewater under high salt conditions and low temperature conditions, and the degradation effect is still excellent in wastewater with multiple polybenzoic acid pollutants coexisting and high concentrations of polybenzoic acid pollutants, and the application range is wide. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 1 is a colony morphology diagram of Pseudomonas putida bacteria in the present invention;

[0019] Figure 2 This is the degradation rate of polybenzoic acid pollutants by Pseudomonas putida;

[0020] Figure 3 is the effect of NaCl concentration on the degradation efficiency of strain PH-20;

[0021] Figure 4 The effect of pH on the degradation efficiency of strain PH-20;

[0022] Figure 5 is the effect of temperature on the degradation efficiency of strain PH-20;

[0023] Figure 6 is the effect of the initial concentration of pyromellitic acid on the degradation efficiency of strain PH-20;

[0024] Figure 7 This is a diagram showing the effect of strain PH-20 in treating actual chemical wastewater. DETAILED DESCRIPTION

[0025] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0026] Example 1, Isolation and identification of strains:

[0027] 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) was added to 100 mL of inorganic salt culture medium containing pyromellitic acid (pyromellitic acid concentration is 200 mg / L). The formula is: NaCl 5.0 g, (NH4)2SO41.0 g, K2HPO4•7H2O 1.96 g, KH2PO40.5 g, MgSO4•7H2O0.2 g, and deionized water is added to 1000 mL, pH 7.0~7.2, sterilized at 121 ℃ for 20 min, and cultured with shaking at 10 ℃. Every 10 days, the culture medium was transferred to fresh inorganic salt culture medium at a 3% inoculum size, and the transfer was repeated 5 times.

[0028] 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 The enriched solution was added into 9.0 mL sterile water and mixed thoroughly to make 10 -2The enrichment solution was serially diluted, and so on. 0.1 mL of each dilution was spread onto an inorganic salt solid medium containing 200 mg / L pyromellitic acid (recipe as above) and incubated at 30°C for 10 days. After 10 days, a single colony was picked from the inorganic salt solid medium and cultured for 24 hours in 3.0 mL of LB liquid medium (recipe: 500 mg / L pyromellitic acid, 10.00 g / L NaCl, 10.00 g / L peptone, 5.00 g / L yeast extract, pH 7.0). The culture was centrifuged at 8000 rpm for 2 minutes, the supernatant was discarded, 3.0 mL of sterile water was added, and the mixture was shaken. The culture was centrifuged again at 8000 rpm for 2 minutes. After washing twice with sterile water, the cells were resuspended in 3.0 mL of sterile water. 1.0 mL of this bacterial solution was added to 100 mL of an inorganic salt liquid medium containing 200 mg / L pyromellitic acid (formula: 200 mg pyromellitic acid, 1.50 g K2HPO4, 0.50 g KH2PO4, 0.20 g MgSO4•7H2O, 1.00 g NaCl, 1.00 g (NH4)2SO4, 20.00 g agar per liter, pH 7.0). The medium was shaken and cultured at 160 rpm and 30°C for 96 hours. The degradation efficiency was then measured by gas chromatography. A strain with the highest degradation efficiency was preserved, namely Pseudomonas putida PH-20 (deposit number: CGMCC No. 33831). Its colony morphology on LB solid medium is as follows: Figure 1 As shown, subsequent experiments were performed.

[0029] The main physiological characteristics of Pseudomonas putida are: rod-shaped, arranged singly or in pairs, approximately 0.5-0.8 μm × 1.5-3.0 μm in size, with terminal flagella, strong motility, and Gram-negative staining. The 16S rRNA gene sequence of Pseudomonas putida is shown in SEQ ID No. 1.

[0030] The above strain was deposited in the General Microbiology Center of China Culture Collection Administration Committee on March 17, 2025, and its classification name is Pseudomonas putida ( Pseudomonas putida ), the strain is named 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.

[0031] Example 2: A method for preparing a microbial agent, comprising the following specific steps:

[0032] S1. Inoculate Pseudomonas putida PH-20 into LB medium (pH 7.0) containing 10.00 g / L NaCl, 10.00 g / L peptone, 5.00 g / L yeast extract, and 1 g / L pyromellitic acid) and culture with shaking until the logarithmic phase.

[0033] S2. Inoculate the cultured bacteria into a seed tank. The culture medium used in the seed tank is as follows: 1 g / L pyromellitic acid, 8 g / L glucose, 1 g / L (NH4)2SO4, 2 g / L K2HPO4, 0.5 g / L MgSO4, 1 g / L NaCl, 0.5 g / L CaCO3, 2 g / L yeast extract, pH 7.2-7.5. Cultivate until the logarithmic growth phase, which is the seed solution.

[0034] S3. The seed liquid is connected to the production tank for fermentation culture. The culture medium used in the production tank is the same as that in the seed tank to obtain a bacterial agent.

[0035] During the cultivation process of steps S2 and S3, the inoculation amount is 8% to 12%, the ventilation rate of sterile air is 1:0.6 to 1.2, the stirring speed is 180 to 240 r / min, the cultivation temperature is 30 to 35°C, and the cultivation time is 48 to 60 h.

[0036] Example 3: Degradation effect of strain PH-20 on polybenzoic acid pollutants:

[0037] Prepare inorganic salt liquid culture media containing terephthalic acid, phthalic acid, isophthalic acid, trimellitic acid, trimesic acid, and pyromellitic acid (initial concentration of each is 500 mg / L). The culture medium formula is: (NH4)2SO4 1.0 g, K2HPO4•7H2O 1.96 g, KH2PO4 0.5 g, MgSO4•7H2O 0.2 g, NaCl concentration is 10 g / L, pH 5.0, and sterilize at 121°C for 20 minutes and let it stand at room temperature. PH-20 seed liquid was added to the culture medium of each pollutant at a 5% inoculum (v / v) and cultured at 35°C and 180 rpm for 96 hours. The content of each pollutant was determined, as shown in the following table. Figure 2 shown.

[0038] Depend on Figure 2 It can be seen that the degradation efficiency of strain PH-20 for terephthalic acid, phthalic acid, isophthalic acid, trimellitic acid, pyromellitic acid and tetracarboxylic acid is above 95%, and the degradation effect is excellent.

[0039] Example 4: Effect of NaCl concentration on the degradation efficiency of strain PH-20:

[0040] The NaCl concentration in the inorganic salt culture medium was adjusted to 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L, and 30 g / L, respectively. Using pyromellitic acid as a model pollutant, 500 mg / L of pyromellitic acid, 1.0 g of (NH4)2SO4, 1.96 g of K2HPO4•7H2O, 0.5 g of KH2PO4, and 0.2 g of MgSO4•7H2O were added to the culture medium, pH 5.0. The culture medium was autoclaved at 121°C for 20 minutes and allowed to cool to room temperature. The seed solution was inoculated into serum bottles at a 5% inoculation rate and incubated in a shaker at 30°C and 180 rpm for 96 hours. The degradation efficiency of pyromellitic acid in the solution was monitored. The results are shown in Table 1. Figure 3 .

[0041] Depend on Figure 3 It can be seen that 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. This shows that strain PH-20 has good tolerance to high salt conditions and is suitable for high-salt chemical wastewater treatment processes.

[0042] Example 5: Effect of pH on the degradation efficiency of strain PH-20:

[0043] Prepare a culture medium containing 500 mg / L pyromellitic acid (pyromellitic acid): (NH4)2SO4 1.0 g, K2HPO4•7H2O 1.96 g, KH2PO4 0.5 g, and MgSO4•7H2O 0.2 g. Autoclave at 121°C for 20 minutes and allow to cool to room temperature. Add PH-20 seed solution to the culture medium containing each contaminant at a 5% inoculum (v / v). Adjust the pH to 4.0, 5.0, 6.0, 7.0, and 8.0, respectively, with 1 mol / L hydrochloric acid and sodium hydroxide. Incubate on a shaker at 30°C and 180 rpm for 96 hours. Determine the contaminant content. Figure 4 shown.

[0044] Depend on Figure 4 It can be seen that the degradation rate of pyromellitic acid by strain PH-20 is above 85% in the pH range of 4.0~8.0, indicating that the strain has strong adaptability to acidity and alkalinity.

[0045] Example 6: Effect of temperature on the degradation efficiency of strain PH-20:

[0046] Prepare a culture medium containing 500 mg / L pyromellitic acid (PBA) with the following components: (NH4)2SO4 1.0 g, K2HPO4•7H2O 1.96 g, KH2PO4 0.5 g, MgSO4•7H2O 0.2 g, pH 5.0. Autoclave at 121°C for 20 minutes and allow to cool to room temperature. Add PH-20 seed solution to the culture medium containing each contaminant at a 5% inoculum (v / v) and incubate at 5°C, 8°C, 15°C, 25°C, and 35°C, at 180 rpm, for 96 hours. Determine the contaminant content, as shown in the following table: Figure 5 shown.

[0047] Depend on Figure 5 It can be seen that the degradation rate of pyromellitic acid by strain PH-20 increases with the increase of temperature in the range of 5~35 ℃. When the degradation temperature is 8~15 ℃, the degradation rate can still reach more than 70%. The results show that strain PH-20 has good low temperature resistance.

[0048] Example 7: Effect of the initial concentration of pyromellitic acid on the degradation efficiency of strain PH-20:

[0049] Prepare culture media with pyromellitic acid concentrations of 0.5, 1, 1.5, 2, 2.5, and 5 g / L, respectively. The culture media components are: (NH4)2SO4 1.0 g, K2HPO4•7H2O 1.96 g, KH2PO4 0.5 g, MgSO4•7H2O 0.2 g, pH 5.0, and sterilize at 121°C for 20 minutes under high pressure. Let it stand at room temperature. Add PH-20 seed solution at a 5% inoculum (v / v) to the culture media of each pollutant. Incubate the culture media at 30°C, 180 rpm, and shake for 96 hours. Determine the pollutant content, as shown in the following table. Figure 6 shown.

[0050] Depend on Figure 6 It can be seen that the degradation rate of pyromellitic acid by strain PH-20 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 degradation effect of strain PH-20 in wastewater containing high concentrations of polybenzoic acid pollutants is also excellent and has a wide range of applications.

[0051] Example 8, application of strain PH-20 in actual chemical wastewater degradation:

[0052] The source of treated water is the wastewater treatment pool of a chemical enterprise with a treatment capacity of 10 L. The bacterial agent prepared by strain PH-20 is added to the sewage at a volume ratio of 5%. The pH of the original sewage is about 6.0 and the temperature is 25 ℃. The initial concentrations of trimellitic acid, pyromellitic acid, pyromellitic acid and COD in the sewage are 1600 mg / L, 1500 mg / L, 1200 mg / L and 5000 mg / L respectively. Aeration treatment is performed to make the dissolved oxygen above 2 mg / L. The concentrations of various pollutants in the wastewater are tested every 12 hours, such as Figure 7 shown.

[0053] Depend on Figure 7 As shown, 72 hours after the addition of strain PH-20, the degradation rates of trimellitic acid, pyromellitic acid, pyromellitic acid, and COD in the wastewater all reached over 90%. These experimental data demonstrate that strain PH-20 is highly effective in degrading polybenzoic acid pollutants in actual chemical wastewater. Furthermore, even when multiple pollutants coexist, strain PH-20 maintains a high degradation rate, suggesting promising application prospects in actual chemical wastewater treatment.

[0054] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any form, and any technical solutions obtained by equivalent replacement or equivalent transformation fall within the scope of protection of the present invention.

Claims

1. A strain of Pseudomonas putida, characterized in that The Pseudomonas putida ( Pseudomonas putida ) was named PH-20 and deposited in the General Microbiology Center of China 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 the chemical wastewater is 0.5-5 g / L, the NaCl concentration is 5-30 g / L, and the temperature is 8-35°C.

3. A bacterial agent produced using the Pseudomonas putida according to claim 1.

4. Use of the bacterial agent according to claim 3 in degrading polybenzoic acid in chemical wastewater, characterized in that: 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 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% to 7% of bacterial agent by volume into chemical wastewater for degradation treatment.

6. A method for preparing the microbial 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. Inoculate the cultured bacteria into a seed tank and culture until the logarithmic growth phase, i.e., the seed solution; S3. The seed liquid is connected to a production tank for fermentation and culture to obtain a bacterial agent.

7. The method for preparing the microbial agent according to claim 6, characterized in that: In step S1, the formula of the LB culture 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 microbial agent according to claim 6, characterized in that: In 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, pH 7.2-7.

5.

9. The method for preparing the microbial 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% to 12%, the ventilation volume of sterile air is 1:0.6 to 1.2, the stirring speed is 180 to 240 r / min, the culture temperature is 30 to 35°C, and the culture time is 48 to 60 h.

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