Pseudomonas aeruginosa NJUXR-CC-3 and application thereof

Abstract

The invention discloses Pseudomonas aeruginosa NJUXR-CC-3 and application of the Pseudomonas aeruginosa NJUXR-CC-3. The Pseudomonas aeruginosa NJUXR-CC-3 is obtained by taking Pseudomonas aeruginosa ATCC 9027 as a starting strain and performing ARTP (Attenuated Region Transfer Protocol) mutagenesis, and the Pseudomonas aeruginosa NJUXR-CC-3 is preserved in the China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC NO: M2025218. The pseudomonas aeruginosa NJUXR-CC-3 can be used for efficiently synthesizing an ampholytic surfactant rhamnolipid by taking vegetable oil as a carbon source; the paraffin inhibition rate of the paraffin inhibitor based on the Pseudomonas aeruginosa is 79.88%, which is 81.59% higher than that of the original strain Pseudomonas aeruginosa ATCC 9027, and the Pseudomonas aeruginosa can be used for preventing pipeline blockage during high-wax crude oil transportation or wax deposition during storage, and has high industrial application value.

Description

Technical Field

[0001] The present invention relates to Pseudomonas aeruginosa, and in particular to a Pseudomonas aeruginosa NJUXR-CC-3 and its applications. Background Art

[0002] Biosurfactants have received extensive attention in recent years in multiple fields due to their unique chemical properties and environmentally friendly characteristics. Rhamnolipid is a typical glycolipid biosurfactant, which is formed by connecting rhamnose and fatty acids through glycosidic bonds. Its hydrophilic group is rhamnose, and its hydrophobic group is a long-chain saturated or unsaturated fatty acid, showing excellent surface activity and biodegradability. It can effectively reduce the surface tension of water and has good emulsifying, solubilizing, wetting, and permeation properties.

[0003] The application fields of rhamnolipid are extensive and continuously expanding. In the field of environmental protection, due to its excellent emulsifying ability, rhamnolipid is widely used in soil remediation and water treatment, especially showing remarkable effects in removing petroleum pollutants and organic pollutants. In the field of clean energy, it can be used in the production of biodiesel and oil-water separation technology to help achieve the efficient development of green energy. In industrial production, as a natural and low-toxic surfactant, rhamnolipid can be used in industries such as textiles, pharmaceuticals, and food processing to improve emulsifying and cleaning effects. In addition, rhamnolipid shows good stability and low irritation in cosmetics and daily chemicals and is suitable as an emulsifier, solubilizer, and moisturizing ingredient.

[0004] Although rhamnolipid has broad application prospects, its industrial production still faces many challenges. Currently, the main production strain of rhamnolipid is Pseudomonas, but problems such as low yield, low substrate conversion rate, and high cost are common in the production process. In addition, the fermentation production of rhamnolipid has high requirements for the stability, adaptability, and metabolic efficiency of the strain, and the performance of existing strains is difficult to meet the industrial demand. Summary of the Invention

[0005] Object of the Invention: The object of the present invention is to provide a novel Pseudomonas aeruginosa NJUXR-CC-3 that efficiently secretes rhamnolipid; the second object is to provide the applications of the Pseudomonas aeruginosa NJUXR-CC-3.

[0006] Technical Solution: The Pseudomonas aeruginosa NJUXR-CC-3 described in the present invention has a deposit number of CCTCC NO: M2025218.

[0007] Deposition Note: Pseudomonas aeruginosa NJUXR-CC-3 in the present invention is deposited in the China Center for Type Culture Collection (CCTCC), with the deposition number CCTCC NO: M2025218 and the deposition date February 13, 2025. The deposition address is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, with the postal code 430064.

[0008] The biological agent described in the present invention contains one or more of Pseudomonas aeruginosa NJUXR-CC-3, its live bacterial culture solution, its freeze-dried live bacteria, and its immobilized live bacteria.

[0009] Application of the Pseudomonas aeruginosa NJUXR-CC-3 or biological agent described in the present invention in the preparation of a paraffin inhibitor.

[0010] Preferably, the application is in the preparation of a paraffin inhibitor for preventing pipeline blockage during the transportation of high-wax crude oil.

[0011] Preferably, the application is in the preparation of a paraffin inhibitor for preventing wax deposition during the storage of high-wax crude oil.

[0012] Preferably, the application is in the preparation of a paraffin inhibitor containing an amphoteric surfactant, and the amphoteric surfactant is rhamnolipid.

[0013] The method for preparing rhamnolipid described in the present invention includes

[0014] (1) Prepare a fermentation medium and ferment using Pseudomonas aeruginosa NJUXR-CC-3 or a biological agent.

[0015] (2) Collect the fermentation broth, centrifuge and collect the supernatant aqueous layer.

[0016] (3) Purify to obtain rhamnolipid.

[0017] Preferably, in step 1, the fermentation medium uses one or more of vegetable oil and waste oil as a carbon source.

[0018] Preferably, in step 1, the fermentation medium uses rapeseed oil as a carbon source.

[0019] Beneficial Effects: Compared with the prior art, the present invention has the following remarkable advantages: 1. The novel Pseudomonas aeruginosa NJUXR-CC-3 can efficiently utilize vegetable oil as a carbon source to produce rhamnolipid; 2. The paraffin inhibition rate of Pseudomonas aeruginosa NJUXR-CC-3 is 79.88%, which is 81.59% higher than that of the original strain Pseudomonas aeruginosa ATCC 9027, and can effectively prevent high-wax crude oil from blocking pipelines or forming wax deposits. Description of the Drawings

[0020] Figure 1It is the rhamnolipid spectrogram in the supernatant of NJUXR-CC-3 fermentation broth. Detailed implementation manners

[0021] The technical solution of the present invention will be further described below.

[0022] Example 1: Mutation and screening of Pseudomonas aeruginosa

[0023] (1) The original strain Pseudomonas aeruginosa ATCC 9027 was taken out from the glycerol tube, streaked on the LB solid medium, and cultured overnight at 37°C.

[0024] (2) A single colony was picked and inoculated into the LB liquid medium, cultured at 37°C for 12 h, sampled, and diluted with the LB liquid medium to OD 600 = 0.8, and glycerol was added to a final concentration of 5% to prepare a bacterial suspension.

[0025] (3) 10 μL of the bacterial suspension obtained in step (2) was pipetted onto a sterile iron sheet, and mutagenesis was performed using the Tianmu Bio Plasma Mutagenesis Breeding Instrument ARTP-M. The voltage was set to 100 V, and the running time was from 60 s to 240 s. After mutagenesis, the iron sheet was placed in 0.5 mL of the LB liquid medium and cultured at 37°C for 30 min. 10 μL of the medium was taken and spread on the LB solid medium, and cultured overnight at 37°C.

[0026] (4) 500 single colonies on the medium were respectively picked and inoculated into a 96-deep well plate with 0.5 mL of the LB liquid medium in each well, and cultured overnight at 37°C and 220 rpm to obtain the seed liquid.

[0027] (5) 100 μL of the seed liquid was inoculated into a 48-square well plate with 2 mL of the fermentation medium in each well. The fermentation medium contained 60 g / L rapeseed oil, 3 g / L yeast extract, 6 g / L NaNO 3 , 1 g / L Na 2 HPO 4 , 1 g / L KH 2 PO 4 , 0.1 g / L MgSO 4 , 0.1 g / L CaCl 2 ·2H2O, pH = 7.0. After inoculation, it was cultured at 37°C and 220 rpm for 48 h.

[0028] (6) The mutant dominant strains were identified by detecting the content of rhamnolipids in the fermentation broth of each bacterium. After 5 rounds of ARTP mutagenesis, 4000 mutant bacteria were obtained. Through high-throughput screening, the Pseudomonas aeruginosa mutant strain NJUXR-CC-3 was finally obtained, and the rhamnolipid spectrum signal in the supernatant of its fermentation broth was as Figure 1 shown. The high-throughput screening method is as follows:

[0029] (61) Take 200 μL of the supernatant of the fermentation broth, add 600 μL of methanol, and centrifuge at 4000 g for 10 min;

[0030] (62) Take 3 μL of the supernatant and add 297 μL of 50% aqueous methanol solution to obtain the test sample;

[0031] (63) Detect the test sample with RapidFire 400, set the mass spectrometry to negative ion mode and SCAN mode, and detect M / Z values of 475.2, 503.2, 529.2, and 531.3.

[0032] Example 2: Preparation of rhamnolipid paraffin inhibitor using Pseudomonas aeruginosa NJUXR-CC-3 with rapeseed oil as the carbon source

[0033] 1. Take out the glycerol bacteria of Pseudomonas aeruginosa NJUXR-CC-3 from the -80 °C refrigerator, inoculate them onto the LB solid medium, and incubate overnight in a 37 °C incubator;

[0034] 2. Pick a single colony of Pseudomonas aeruginosa NJUXR-CC-3 from the plate and inoculate it into a test tube containing 3 mL of LB liquid medium. Incubate it on a shaker at 37 °C and 200 rpm for 16 h to obtain the primary seed solution;

[0035] 3. Inoculate 2 mL of the primary seed solution into a 250 mL conical flask containing 50 mL of LB liquid medium and incubate it on a shaker at 37 °C and 200 rpm for 16 h to obtain the secondary seed solution;

[0036] 4. Inoculate 33 mL of the secondary seed solution into a 2 L conical flask containing 1 L of fermentation medium and incubate it on a shaker at 37 °C and 200 rpm for 48 h. The fermentation medium contains 60 g / L rapeseed oil, 3 g / L yeast extract, 6 g / L NaNO 3 ,1 g / L Na 2 HPO 4 ,1 g / L KH 2 PO 4 ,0.1 g / L MgSO 4 ,0.1 g / L CaCl 2 ·2H 2 O, pH 7.0;

[0037] 5. After the cultivation is completed, collect the fermentation broth, centrifuge at 9000 rpm for 10 min, and take the supernatant aqueous layer as the paraffin inhibitor cleaner.

[0038] Comparative Example 1: Preparation of rhamnolipid paraffin inhibitor using Pseudomonas aeruginosa ATCC 9027 with rapeseed oil as the carbon source

[0039] 1. Take out the Pseudomonas aeruginosa ATCC 9027 glycerol bacteria from the -80°C refrigerator, inoculate them onto the LB solid medium, and culture them overnight in a 37°C incubator;

[0040] 2. Pick a single colony of Pseudomonas aeruginosa ATCC 9027 from the plate, inoculate it into a test tube containing 3 mL of LB liquid medium, and culture it on a shaker at 37°C and 200 rpm for 16 h to obtain the primary seed liquid;

[0041] 3. Inoculate 2 mL of the primary seed liquid into a 250 mL conical flask containing 50 mL of LB liquid medium, and culture it on a shaker at 37°C and 200 rpm for 16 h to obtain the secondary seed liquid;

[0042] 4. Inoculate 33 mL of the secondary seed liquid into a 2 L conical flask containing 1 L of fermentation medium, and culture it on a shaker at 37°C and 200 rpm for 48 h. The fermentation medium contains 60 g / L rapeseed oil, 3 g / L yeast extract, 6 g / L NaNO 3 、1 g / L Na 2 HPO 4 、1 g / L KH 2 PO 4 、0.1 g / L MgSO 4 、0.1 g / L CaCl 2 ·2H 2 O, pH 7.0;

[0043] 5. After the culture is completed, collect the fermentation broth, centrifuge it at 9000 rpm for 10 min, and take the supernatant aqueous layer as the paraffin prevention cleaner. Comparative Example 2: Preparation of rhamnolipid paraffin inhibitor using Pseudomonas aeruginosa NJUXR-CC-3 with soybean oil as the carbon source

[0044] 1. Take out the Pseudomonas aeruginosa NJUXR-CC-3 glycerol bacteria from the -80°C refrigerator, inoculate them onto the LB solid medium, and culture them overnight in a 37°C incubator;

[0045] 2. Pick a single colony of Pseudomonas aeruginosa NJUXR-CC-3 from the plate, inoculate it into a test tube containing 3 mL of LB liquid medium, and culture it on a shaker at 37°C and 200 rpm for 16 h to obtain the primary seed liquid;

[0046] 3. Inoculate 2 mL of the primary seed liquid into a 250 mL conical flask containing 50 mL of LB liquid medium, and culture it on a shaker at 37°C and 200 rpm for 16 h to obtain the secondary seed liquid;

[0047] 4. Inoculate 33 mL of the secondary seed liquid into a 2-L conical flask containing 1 L of the fermentation medium, and culture it in a shaker at 37 °C and 200 rpm for 48 h. The fermentation medium contains 60 g / L soybean oil, 3 g / L yeast extract, 6 g / L NaNO 3 、1 g / L Na 2 HPO 4 、1 g / L KH 2 PO 4 、0.1 g / L MgSO 4 、0.1 g / L CaCl 2 ·2H 2 O, pH 7.0;

[0048] 5. After the cultivation is completed, collect the fermentation broth, centrifuge it at 9000 rpm for 10 min, and take the supernatant aqueous layer as the paraffin inhibitor cleaner. Comparative Example 3: Pseudomonas aeruginosa NJUXR-CC-3 uses corn oil as a carbon source to prepare a rhamnolipid paraffin inhibitor

[0049] 1. Take out the glycerol bacteria of Pseudomonas aeruginosa NJUXR-CC-3 from the -80 °C refrigerator, inoculate them into the LB solid medium, and culture them overnight in a 37 °C incubator;

[0050] 2. Pick a single colony of Pseudomonas aeruginosa NJUXR-CC-3 from the plate and inoculate it into a test tube containing 3 mL of LB liquid medium. Culture it in a shaker at 37 °C and 200 rpm for 16 h to obtain the primary seed liquid;

[0051] 3. Inoculate 2 mL of the primary seed liquid into a 250-mL conical flask containing 50 mL of LB liquid medium, and culture it in a shaker at 37 °C and 200 rpm for 16 h to obtain the secondary seed liquid;

[0052] 4. Inoculate 33 mL of the secondary seed liquid into a 2-L conical flask containing 1 L of the fermentation medium, and culture it in a shaker at 37 °C and 200 rpm for 48 h. The fermentation medium contains 60 g / L corn oil, 3 g / L yeast extract, 6 g / L NaNO 3 、1 g / L Na 2 HPO 4 、1 g / L KH 2 PO 4 、0.1 g / L MgSO 4 、0.1 g / L CaCl 2 ·2H 2 O, pH 7.0;

[0053] 5. After the cultivation is completed, collect the fermentation broth, centrifuge it at 9000 rpm for 10 min, and take the supernatant aqueous layer as the paraffin inhibitor cleaner.

[0054] Test Example: Determination of Paraffin Inhibition Rate of Paraffin Inhibitors in Examples and Comparative Examples

[0055] 1. Take 9 clean and dry 30 mL glass test tubes (18×180 mm), and label them as tubes No. 1 - 15. Add samples of B2 crude oil (provided by CNOOC Tianjin Chemical Research and Design Institute) to each tube, and precisely weigh the mass of the test tubes;

[0056] 2. Experimental group: Add 4 mL of the paraffin inhibitor prepared in Comparative Example 1 to tubes No. 1 - 3, add 4 mL of the paraffin inhibitor prepared in Comparative Example 2 to tubes No. 4 - 6, add 4 mL of the paraffin inhibitor prepared in Comparative Example 3 to tubes No. 7 - 9, and add 4 mL of the paraffin inhibitor prepared in Example 2 to tubes No. 10 - 12; Control group: Add 4 mL of sterile water to tubes No. 13 - 15;

[0057] 3. After incubating the test tubes in a 60°C water bath incubator for 1 h, seal the test tubes and place them in a shaker at 37°C and 220 rpm for 96 h;

[0058] 4. Invert the test tubes, discard the flowing liquid, and continue to invert them in a 45°C shaker overnight to achieve the purpose of drying. At this time, its mass remains basically unchanged, which is the paraffin - wax - formed tube of the crude oil;

[0059] 5. Precisely weigh the mass of the paraffin - wax - formed tubes, and subtract the mass of the empty test tubes respectively to obtain the paraffin - wax mass in each tube and calculate the average paraffin - wax mass. The calculation method of the paraffin inhibition rate is the ratio of the difference between the average paraffin - wax masses of the control group and the experimental group to the average paraffin - wax mass of the control group.

[0060] The results are shown in Table 1. The paraffin inhibition rate of the paraffin inhibitor prepared using Pseudomonas aeruginosa NJUXR - CC - 3 is 79.88%, which is significantly higher than 43.99% of the paraffin inhibitor prepared using Pseudomonas aeruginosa ATCC 9027. The paraffin inhibition effect is increased by 81.59%, and it can effectively prevent high - wax crude oil from blocking pipelines or forming wax deposits.

[0061] Table 1 Paraffin Inhibition Rates of Pseudomonas aeruginosa ATCC 9027 (WT) and NJUXR - CC - 3

[0062]

Claims

1. A Pseudomonas aeruginosa NJUXR-CC-3, whose deposit number is CCTCCNO: M2025218.

2. A biological agent, characterized in that: The biological preparation contains one or more of Pseudomonas aeruginosa NJUXR-CC-3, its live bacteria culture solution, its live bacteria freeze-dried powder, and its immobilized live bacteria.

3. Use of the Pseudomonas aeruginosa NJUXR-CC-3 according to claim 1 or the biological preparation according to claim 2 in the preparation of a wax inhibitor.

4. The use according to claim 3, characterized in that: The application is the application in preparing a wax inhibitor for preventing pipelines from being blocked during transportation of high-wax crude oil.

5. The use according to claim 3, characterized in that: The application is application in preparing a wax inhibitor for preventing wax deposition during storage of high-wax crude oil.

6. The use according to any one of claims 3 to 5, characterized in that: The application is application in preparing a wax inhibitor containing an amphoteric surfactant.

7. The use according to claim 6, characterized in that: The amphoteric surfactant is rhamnolipid.

8. A method for preparing rhamnolipid, characterized in that: include (1) preparing a fermentation medium and using Pseudomonas aeruginosa NJUXR-CC-3 or a biological preparation for fermentation; (2) collecting the fermentation broth, centrifuging and collecting the supernatant aqueous phase; (3) Purify and obtain rhamnolipid.

9. The method for preparing rhamnolipid according to claim 8, characterized in that: In step 1, the fermentation medium uses one or more of vegetable oil and waste cooking oil as a carbon source.

10. The method for preparing rhamnolipid according to claim 9, characterized in that: In the step 1, the fermentation medium uses rapeseed oil as a carbon source.

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