Pseudomonas aeruginosa and application of fermentation product of the same

Abstract

This invention belongs to the field of biotechnology, specifically relating to a strain of *Pseudomonas aeruginosa*, and further disclosing the application of its fermentation product of medium-chain dirhamnolipids. The *Pseudomonas aeruginosa* PA001 obtained through screening can ferment high yields of medium-chain dirhamnolipids using glycerol as a raw material under optimized culture conditions. Its fermentation product not only exhibits excellent emulsifying properties for petroleum (with an emulsification coefficient reaching 55%), but also has a petroleum solubilizing effect, increasing the solubility of petroleum in aqueous phase by more than 3.3 times, demonstrating promising application prospects in the petrochemical field.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a Pseudomonas aeruginosa strain, and further disclosing the application of its fermentation product, a medium-chain dirhamnolipid. Background Technology

[0002] As a non-renewable energy source, oil recovery is an urgent task. As early as 1957, researchers injected *Pseudomonas aeruginosa* along with other substances into oil reservoirs and verified that this improved oil recovery efficiency. Studies show that microbial enhanced oil recovery (MEOR) technology mainly relies on specific microorganisms and their metabolites. Biosurfactants are the main mechanism of microbial enhanced oil recovery, reducing the tension at the oil-water interface and the adhesion of oil, thus significantly improving oil extraction efficiency. In field applications, to further reduce costs, microorganisms or microbial fermentation broth can be directly injected into the oil reservoir. According to literature reports, Daqing Oilfield conducted research on the improvement of oil recovery using biosurfactants and chemical surfactants, finding that rhamnolipids significantly improve recovery while significantly reducing oil production costs compared to chemical surfactants.

[0003] Rhamnolipids are a class of anionic biosurfactants produced by microorganisms, possessing excellent detergency, emulsification, solubilization, wetting, and penetration properties. Compared with traditional chemical surfactants, rhamnolipids have advantages such as non-toxicity, antibacterial properties, strong emulsifying ability, and biodegradability, showing great promise for applications in bioenergy, petrochemicals, biopharmaceuticals, food, and daily chemicals. They are currently among the most well-researched biosurfactants.

[0004] Currently, rhamnolipids are mainly synthesized by microbial metabolism, with *Pseudomonas aeruginosa* being the most commonly used microorganism for rhamnolipid production due to its high yield of glycolipid products. Studies show that rhamnolipids produced by *P. aeruginosa* fermentation are mainly composed of a series of homologues of monorhamnolipids and dirhamnolipids, among which dirhamnolipids have higher emulsifying capabilities, which are more significant in applications such as crude oil emulsification. During the fermentation process of rhamnolipid production, the composition of the glycolipid products is comprehensively affected by a variety of complex factors, including the microbial strain, culture medium composition, and fermentation conditions. Based on literature review, compared to using glycerol as a raw material, rhamnolipids fermented from various oils often have complex and diverse product compositions, posing certain difficulties for subsequent product extraction and purification, and hindering the acquisition of rhamnolipids of consistent quality. Therefore, in order to fully utilize the advantages of rhamnolipids in petroleum extraction, it is necessary to further optimize the fermentation process and obtain microbial strains and fermentation methods with dirhamnolipids as the main product and a concentrated distribution, which is of positive significance for the development of petroleum extraction technology. Summary of the Invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a Pseudomonas aeruginosa PA001 strain that can ferment high-yield medium-chain dirhamnolipids using glycerol as raw material under optimized culture conditions. It has good emulsifying and solubilizing effects on petroleum and has good application prospects in the petrochemical field.

[0006] The second technical problem to be solved by the present invention is to provide the application of the above-mentioned Pseudomonas aeruginosa PA001 in the fermentation production of carbon-chain dirhamnolipids.

[0007] To solve the above-mentioned technical problems, the present invention provides a Pseudomonas aeruginosa strain, classified as Pseudomonas aeruginosa PA001, which has been deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 31150.

[0008] The present invention also provides a method for fermenting and producing rhamnolipids, comprising the step of inoculating the Pseudomonas aeruginosa strain into a suitable fermentation medium for fermentation culture.

[0009] Specifically, in the method for producing rhamnolipids by fermentation, the fermentation medium comprises the following components in the indicated mass percentages: 2-5 wt% carbon source, 0.5-2 wt% nitrogen source, 0.1-1.0 wt% first inorganic salt, 0.1-1.0 wt% second inorganic salt, 0.01-0.1 wt% metal ions, and pH 6.8-7.2.

[0010] The first inorganic salt is a phosphate.

[0011] Specifically, the method for producing rhamnolipids through fermentation:

[0012] The carbon source includes glycerol; and / or,

[0013] The nitrogen source includes at least one selected from sodium nitrate, ammonium chloride, urea, ammonium sulfate, peptone, diammonium hydrogen phosphate, or yeast extract; preferably, the nitrogen source includes sodium nitrate; and / or,

[0014] The phosphate includes at least one of disodium hydrogen phosphate or potassium dihydrogen phosphate; and / or,

[0015] The metal ions include Mg 2+ Ca 2+ or Fe 2+ At least one of them.

[0016] Specifically, in the method for fermenting rhamnolipids, the fermentation medium comprises the following components in the indicated mass percentages: glycerol 3-5 wt%, NaNO3 0.8-1.0 wt%, yeast extract 0.3-0.8 wt%, NaCl 0.05-0.2 wt%, KCl 0.05-0.2 wt%, Na2HPO4 0.1-0.2 wt%, and KH2PO4.

[0017] 0.01-0.1wt%, MgSO4·7H2O 0.01-0.03wt%, CaCl2·2H2O 0.005-0.02wt%.

[0018] Preferably, the fermentation medium comprises the following components in the indicated mass percentages: 4 wt% glycerol, 0.9 wt% NaNO3, 0.5 wt% yeast extract, 0.1 wt% NaCl, 0.1 wt% KCl, 0.15 wt% Na2HPO4, 0.05 wt% KH2PO4, 0.02 wt% MgSO4·7H2O, and 0.01 wt% CaCl2·2H2O.

[0019] Specifically, the fermentation production method for rhamnolipids includes the following fermentation conditions: controlling the pH of the fermentation medium to 6.0-7.0 and fermenting at 30℃-37℃ for 72h-120h.

[0020] Specifically, the method for producing rhamnolipid by fermentation further includes the step of inoculating the Pseudomonas aeruginosa strain of claim 1 into a seed culture medium for seed liquid culture;

[0021] Preferably, the conditions for seed culture include: controlling the rotation speed at 200-250 r / min and culturing at 35-40℃ for 12-24 h.

[0022] Specifically, the method for producing rhamnolipin by fermentation also includes the step of collecting the fermentation products for rhamnolipin extraction;

[0023] The extraction step includes centrifugation, calcium precipitation, and refolding of the fermentation product.

[0024] The present invention also discloses the application of the aforementioned Pseudomonas aeruginosa in the fermentation production of rhamnolipids, especially medium-chain dirhamnolipids.

[0025] The present invention also discloses the application of the aforementioned Pseudomonas aeruginosa and its fermentation products in the petroleum field, particularly in the preparation of petroleum additives;

[0026] Preferably, the petroleum additive includes surfactants, petroleum solubilizers, or petroleum emulsifiers.

[0027] The Pseudomonas aeruginosa PA001 provided by this invention has the characteristics and properties of high-yield medium-chain dirhamnolipids fermented from glycerol. More than 80% of the rhamnolipid products obtained by its fermentation are medium-chain dirhamnolipids, and the products are concentrated.

[0028] The Pseudomonas aeruginosa PA001 strain obtained through screening in this invention can ferment high-yield medium-chain dirhamnolipids using glycerol as a raw material under optimized culture conditions. Its fermentation products not only have a good emulsifying effect on petroleum, with an emulsification coefficient of up to 55%, but also have the effect of solubilizing petroleum, increasing the solubility of petroleum in the aqueous phase by more than 3.3 times. It has good application prospects in the petrochemical field. Attached Figure Description

[0029] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein...

[0030] Figure 1 The growth curve of strain PA001 described in this invention in seed culture medium;

[0031] Figure 2 The effect of the nitrogen source described in this invention on the production of rhamnolipids by strain PA001;

[0032] Figure 3 The fermentation process of strain PA001 described in this invention in a 5L fermenter. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention.

[0034] Example 1

[0035] In this embodiment, the soil around the centralized kitchen waste treatment site was initially screened, and the strains were obtained by secondary screening using CTAB-methylene blue plate screening combined with the oil drainage ring method.

[0036] Upon identification, the colony morphology of the strain was as follows: grayish-green colonies with irregular edges, smooth and opaque surface, moist, and easily picked up. The bacterial cell morphology was as follows: Gram-negative, short rod-shaped, and non-spore-forming. The physiological and biochemical characteristics of the strain were as follows: aerobic, positive for catalase, negative for methylation, positive for indole, and positive for citrate utilization.

[0037] The strain screened in this embodiment was identified as Pseudomonas aeruginosa, and its classification name is Pseudomonas aeruginosa PA001. It is deposited at the China General Microbiological Culture Collection Center on July 3, 2024, with accession number CGMCC No. 31150.

[0038] Verification showed that the Pseudomonas aeruginosa PA001 strain screened in this embodiment has the characteristic of high-yield of medium-chain dirhamnolipids fermented from glycerol. More than 80% of its rhamnolipid fermentation products are medium-chain dirhamnolipids, and the products are concentrated.

[0039] Example 2

[0040] In this embodiment, the method for producing rhamnolipids by fermentation using Pseudomonas aeruginosa PA001 includes the following steps:

[0041] (1) Seed preparation: After thawing the bacterial culture stored at -80℃, it was streaked onto LB solid medium and incubated upside down in a 37℃ constant temperature incubator for activation; a single colony was picked from the activated plate and inoculated into LB liquid medium, and cultured overnight at 37℃ and 220 r / min to obtain the seed culture; in this embodiment, the growth curve of the PA001 strain in the seed culture medium is shown in the attached figure. Figure 1 As shown;

[0042] (2) Fermentation culture: The seed liquid obtained in step (1) was inoculated into the fermentation medium at an inoculation rate of 5%, the pH of the fermentation medium was controlled at 6.5, and the culture was carried out at 35℃ for 96 hours to obtain the fermentation liquid.

[0043] In this embodiment, the fermentation medium formula is as follows (g / L): glycerol 40g / L, NaNO3 9g / L, yeast extract 5g / L, NaCl 1g / L, KCl 1g / L, Na2HPO4 1.5g / L, KH2PO4 0.5g / L, MgSO4·7H2O 0.2g / L, CaCl2·2H2O 0.1g / L;

[0044] (3) Product extraction: The fermentation broth obtained in step (2) was used to extract rhamnolipin. The fermentation broth was centrifuged to obtain the supernatant. The pH of the supernatant was adjusted to 8.0. After heating to 50°C, an equal volume of saturated CaCl2 solution was slowly added under vigorous stirring so that the mass ratio of CaCl2 to rhamnolipin was 1:2. Samples were taken every 1 hour of reaction. After 2 hours of reaction, the supernatant was centrifuged at 15000 rpm for 3 minutes. The concentration of rhamnolipin (calcium) was determined by the sulfuric acid anthrone method, and the yield was calculated. The precipitate was further washed thoroughly with NaOH solution at pH 9.0 and centrifuged at 15000 rpm for 3 minutes. The precipitate was weighed and dissolved in acetic acid and the concentration was determined to obtain rhamnolipin.

[0045] Example 3

[0046] In this embodiment, the method for producing rhamnolipids by fermentation using Pseudomonas aeruginosa PA001 includes the following steps:

[0047] (1) Seed preparation: After thawing the bacterial culture stored at -80℃, it was streaked onto LB solid medium and incubated upside down in a 37℃ constant temperature incubator for activation; single colonies were picked from the activated plate and inoculated into LB liquid medium and incubated overnight at 30℃ and 250r / min to obtain seed culture;

[0048] (2) Fermentation culture: The seed liquid obtained in step (1) was inoculated into the fermentation medium at an inoculation rate of 3%, the pH of the fermentation medium was controlled at 6.0, and the culture was carried out at 37℃ for 72h to obtain the fermentation liquid.

[0049] In this embodiment, the fermentation medium formula is as follows (g / L): glycerol 30g / L, NaNO3 10g / L, yeast extract 3g / L, NaCl 2g / L, KCl 0.5g / L, Na2HPO4 2g / L, KH2PO4 0.1g / L, MgSO4·7H2O 0.3g / L, CaCl2·2H2O 0.05g / L;

[0050] (3) Product extraction: The fermentation broth obtained in step (2) was used to extract rhamnolipin. The fermentation broth was centrifuged to obtain the supernatant. The pH of the supernatant was adjusted to 8.0. After heating to 50°C, an equal volume of saturated CaCl2 solution was slowly added under vigorous stirring so that the mass ratio of CaCl2 to rhamnolipin was 1:2. Samples were taken every 1 hour of reaction. After 2 hours of reaction, the supernatant was centrifuged at 15000 rpm for 3 minutes. The concentration of rhamnolipin (calcium) was determined by the sulfuric acid anthrone method, and the yield was calculated. The precipitate was further washed thoroughly with NaOH solution at pH 9.0 and centrifuged at 15000 rpm for 3 minutes. The precipitate was weighed and dissolved in acetic acid and the concentration was determined to obtain rhamnolipin.

[0051] Example 4

[0052] In this embodiment, the method for producing rhamnolipids by fermentation using Pseudomonas aeruginosa PA001 includes the following steps:

[0053] (1) Seed preparation: After thawing the bacterial culture stored at -80℃, it was streaked onto LB solid medium and incubated upside down in a 37℃ constant temperature incubator for activation; single colonies were picked from the activated plate and inoculated into LB liquid medium and incubated overnight at 37℃ and 200r / min to obtain seed culture;

[0054] (2) Fermentation culture: The seed liquid obtained in step (1) was inoculated into the fermentation medium at an inoculation rate of 2%, the pH of the fermentation medium was controlled at 7.0, and the culture was carried out at 30℃ for 120h to obtain the fermentation liquid.

[0055] In this embodiment, the fermentation medium formula is as follows (g / L): glycerol 50g / L, NaNO3 8g / L, yeast extract 8g / L, NaCl 0.5g / L, KCl 2g / L, Na2HPO4 1g / L, KH2PO4 1g / L, MgSO4·7H2O 0.1g / L, CaCl2·2H2O 0.2g / L;

[0056] (3) Product extraction: The fermentation broth obtained in step (2) was used to extract rhamnolipin. The fermentation broth was centrifuged to obtain the supernatant. The pH of the supernatant was adjusted to 8.0. After heating to 50°C, an equal volume of saturated CaCl2 solution was slowly added under vigorous stirring so that the mass ratio of CaCl2 to rhamnolipin was 1:2. Samples were taken every 1 hour of reaction. After 2 hours of reaction, the supernatant was centrifuged at 15000 rpm for 3 minutes. The concentration of rhamnolipin (calcium) was determined by the sulfuric acid anthrone method, and the yield was calculated. The precipitate was further washed thoroughly with NaOH solution at pH 9.0 and centrifuged at 15000 rpm for 3 minutes. The precipitate was weighed and dissolved in acetic acid and the concentration was determined to obtain rhamnolipin.

[0057] Example 5

[0058] This embodiment, based on the scheme of Example 2, investigates the effects of different nitrogen sources on the production of rhamnolipids by strain PA001. Specifically, it replaces equal amounts of sodium nitrate and yeast extract with different nitrogen sources. The results of the effects of different nitrogen source systems on the production of rhamnolipids by strain PA001 are attached. Figure 2 .

[0059] Example 6

[0060] This embodiment, based on the scheme of Embodiment 2, verifies the effect of strain PA001 in a 5L fermenter. The fermentation process of strain PA001 in the 5L fermenter in this embodiment is as follows: Figure 3As shown.

[0061] Experimental Example

[0062] 1. Product testing

[0063] In this experimental example, the components of the fermentation products in Examples 2-4 above were detected, and the results are shown in Table 1 below. The table only shows the main components (accounting for more than 90% of the total components) in terms of glycolipid type, and the content is the total content of each component.

[0064] Table 1. Composition of fermentation products

[0065]

[0066]

[0067] It is evident that the strain PA001 screened in this invention can synthesize medium-chain dirhamnolipids relatively well.

[0068] 2. Emulsified petroleum

[0069] In this experimental example, the products prepared in Examples 2-4 above were used to verify the emulsification performance of liquid paraffin.

[0070] Add 1.5 mL of fermentation supernatant to a graduated test tube, then add 0.9 g of liquid paraffin, shake manually 100 times, let stand for 24 h, observe the emulsifying activity, and further determine and calculate the E24 value; the E24 calculation formula is as follows:

[0071] E24 = (Emulsion layer height / Total liquid height) × 100%.

[0072] The results are shown in Table 2 below.

[0073] Table 2 Emulsification Performance Results

[0074] Example Rhamnose lipid yield (g / L) Emulsifying properties E24 (%) Example 2 8.35±0.23 76.76 Example 3 7.83±0.04 64.55 Example 4 7.23±0.00 58.11

[0075] It is evident that the fermentation product of the PA001 strain described in this invention exhibits good petroleum emulsification properties.

[0076] In summary, the Pseudomonas aeruginosa PA001 obtained through screening in this invention can ferment high-yield medium-chain dirhamnolipids using glycerol as a raw material under optimized culture conditions. Its fermentation products not only have a good emulsifying effect on petroleum, with an emulsification coefficient of up to 55%, but also have the effect of solubilizing petroleum, increasing the solubility of petroleum in the aqueous phase by more than 3.3 times. It has good application prospects in the petrochemical field.

[0077] The embodiments of the present invention have been described in detail above. Specific examples have been used to illustrate the principles and implementation methods of the present invention. The description of the above embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of the present invention. Therefore, the content of this specification should not be construed as a limitation of the present invention.

Claims

1. A Pseudomonas aeruginosa, characterized in that, Its classification name is Pseudomonas aeruginosa PA001, and it has been deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 31150.

2. A method for the fermentative production of rhamnolipids, characterized in that, The method includes the step of inoculating the *Pseudomonas aeruginosa* strain of claim 1 into a suitable fermentation medium for fermentation culture.

3. The process for the fermentative production of rhamnolipids according to claim 2, characterized in that, The fermentation medium comprises the following components in the indicated mass percentages: 2-5 wt% carbon source, 0.5-2 wt% nitrogen source, 0.1-1.0 wt% first inorganic salt, 0.1-1.0 wt% second inorganic salt, and 0.01-0.1 wt% metal ions. pH 6.8-7.2; The first inorganic salt is a phosphate.

4. The method for producing rhamnolipids by fermentation according to claim 3, characterized in that: The carbon source includes glycerol; and / or, The nitrogen source includes at least one of sodium nitrate, ammonium chloride, urea, ammonium sulfate, peptone, diammonium hydrogen phosphate, or yeast extract; and / or, The phosphate includes at least one of disodium hydrogen phosphate or potassium dihydrogen phosphate; and / or, The metal ions include at least one of Mg 2+ , Ca 2+ , or Fe 2+ .

5. The process for the fermentative production of rhamnolipids according to any one of claims 2 to 4, characterized in that, The fermentation medium comprises the following components in the indicated mass percentages: 3-5 wt% glycerol, NaNO3 0.8-1.0 wt%, yeast extract 0.3-0.8 wt%, NaCl 0.05-0.2 wt%, KCl 0.05-0.2 wt%, Na2HPO4 0.1-0.2 wt%, KH2PO4 0.01-0.1 wt%, MgSO4·7H2O 0.01-0.03 wt%, CaCl2·2H2O 0.005-0.02 wt%; Preferably, the fermentation medium comprises the following components in the indicated mass percentages: 4 wt% glycerol, 0.9 wt% NaNO3, 0.5 wt% yeast extract, 0.1 wt% NaCl, 0.1 wt% KCl, 0.15 wt% Na2HPO4, 0.05 wt% KH2PO4, 0.02 wt% MgSO4·7H2O, and 0.01 wt% CaCl2·2H2O.

6. The method for producing rhamnolipids by fermentation according to any one of claims 2-5, characterized in that, The fermentation conditions include: controlling the pH of the fermentation medium to 6.0-7.0, and fermenting at 30℃-37℃ for 72h-120h.

7. The method for producing rhamnolipids by fermentation according to any one of claims 2-6, characterized in that, It also includes the step of inoculating the Pseudomonas aeruginosa strain of claim 1 into a seed culture medium for seed liquid culture; Preferably, the conditions for seed culture include: controlling the rotation speed at 200-250 r / min and culturing at 35-40℃ for 12-24 h.

8. The method for producing rhamnolipids by fermentation according to any one of claims 2-7, characterized in that, It also includes the step of collecting fermentation products for rhamnolipid extraction; The extraction step includes centrifugation, calcium precipitation, and refolding of the fermentation product.

9. The application of the *Pseudomonas aeruginosa* of claim 1 in the fermentation production of rhamnolipids, especially medium-chain dirhamnolipids.

10. The application of the *Pseudomonas aeruginosa* and its fermentation products as described in claim 1, particularly in the petroleum industry, for the preparation of petroleum additives; Preferably, the petroleum additive includes surfactants, petroleum solubilizers, or petroleum emulsifiers.

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