Pseudomonas aeruginosa and application thereof

Abstract

The invention belongs to the technical field of microbial fermentation, and particularly relates to pseudomonas aeruginosa and application thereof. According to the specific technical scheme, the pseudomonas aeruginosa HY-8 is preserved in the Guangdong Microbial Culture Collection Center on October 24, 2024, and the preservation number of the pseudomonas aeruginosa HY-8 is GDMCC (China General Microbiological Culture Collection Center) NO: 65339. The invention provides a strain of pseudomonas aeruginosa which can tolerate high-concentration ethanol and can produce rhamnolipid at high yield. The pseudomonas aeruginosa is used for producing the rhamnolipid through fermentation, and ethanol is added in the fermentation process, so that the problem that a large amount of foam is generated in the fermentation process can be effectively solved, the normal growth of the pseudomonas aeruginosa cannot be influenced, and the continuity and stability of the rhamnolipid produced through fermentation can be ensured, thereby greatly improving the yield of the rhamnolipid produced through fermentation.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and specifically relates to a strain of Pseudomonas aeruginosa and application thereof. Background Art

[0002] Rhamnolipid is a natural polysaccharide with multiple physiological activities. It can be antioxidant, anti-inflammatory, antiviral, antibacterial, and anti-thrombotic. It can also be used as a drug carrier and has broad application prospects in the fields of medicine, food, and cosmetics.

[0003] Some microorganisms have the ability to secrete rhamnolipids. Common microorganisms that can produce rhamnolipids include Pseudomonas, Klebsiella, Acinetobacter and Lactobacillus. However, there are obvious technical defects in the production of rhamnolipids by microbial fermentation, which limits its industrial application. On the one hand, excessively high concentrations of substrates in the fermentation broth are toxic to bacterial cells, so the concentration of the added substrate cannot be too high, resulting in low conversion efficiency. On the other hand, a large amount of foam will be generated during the fermentation process. If it is not effectively controlled, the fermentation broth will all bubble up within 24 hours. The main reasons for the generation of foam include: ventilation and stirring are required during fermentation, the air is mechanically dispersed and foam is formed; there are colloidal substances such as protein, sugar and fat in the fermentation broth, which will further promote the formation of foam; in addition, the overall environment of the fermentation broth (temperature, concentration and pH factors) will also promote the generation of foam, for example, high concentration and high temperature conditions will increase the stability of the foam.

[0004] These foams will seriously reduce the fermentation efficiency, mainly because: (1) The persistent presence of foam will hinder the removal of carbon dioxide, affect the absorption and utilization of oxygen by microorganisms, destroy normal physiological metabolism, and be detrimental to fermentation and biosynthesis. (2) The presence of foam will greatly reduce the effective volume of the fermentation tank and affect the utilization rate of the equipment. In the prior art, the liquid volume in the fermentation tank is generally only about 50%. (3) Excessive foam may cause a large amount of material to escape, resulting in waste and environmental pollution. (4) The foam may rise to the top of the tank and seep out from the shaft seal, increasing the chance of contamination. (5) Excessive foam will also affect oxygen transfer, ventilation and stirring effects, leading to abnormal fermentation and further reducing production.

[0005] Therefore, the key to solve the foam problem during fermentation is to use microbial fermentation to produce rhamnolipids. In the prior art, the general treatment method is to add a defoamer. However, the defoaming effect of common mineral oil defoamers on the market is not significant. Only silicone defoamers can reduce the amount of foam, but their effect is not ideal, and the effect can only last for a few minutes after addition. Therefore, a large amount of defoamers are often required to ferment a batch of rhamnolipids, which not only greatly increases the fermentation cost, but also brings great difficulties to the downstream separation and purification.

[0006] In summary, if a new microorganism with high rhamnolipid production can be provided and the problem of large-scale foam generation during fermentation can be solved, it will have great application potential. Summary of the invention

[0007] The purpose of the invention is to provide a strain of Pseudomonas aeruginosa and application thereof.

[0008] To achieve the above-mentioned purpose of the invention, the technical solution adopted by the present invention is: a strain of Pseudomonas aeruginosa (Pseudomonas aeruginosa) HY-8 was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 24, 2024, with a deposit number of: GDMCC NO: 65339.

[0009] Correspondingly, a strain of Pseudomonas aeruginosa, the 16S rDNA sequence of the Pseudomonas aeruginosa is shown as SEQ ID NO:1.

[0010] Accordingly, a bacterial preparation is prepared using the Pseudomonas aeruginosa.

[0011] Correspondingly, a bacterial preparation comprising the Pseudomonas aeruginosa.

[0012] Correspondingly, a method for preparing rhamnolipid using the Pseudomonas aeruginosa or the bacterial preparation.

[0013] Preferably, the method comprises: utilizing the Pseudomonas aeruginosa to ferment and produce rhamnolipid.

[0014] Preferably, ethanol is added to the fermentation system.

[0015] Preferably, vegetable oil and / or glycerol is used as a carbon source during fermentation, for example, rapeseed oil is used as a carbon source.

[0016] Preferably, the fermentation temperature is 28-35°C; the fermentation pH is 6-8.

[0017] The present invention has the following beneficial effects: the present invention provides a new strain of Pseudomonas aeruginosa, whose ethanol tolerance is more than 10 times that of ordinary Pseudomonas aeruginosa, and can produce high rhamnolipids. Based on this characteristic of the Pseudomonas aeruginosa, the present invention further provides a method for producing high rhamnolipids. Adding part of ethanol during the fermentation process can effectively solve the problem of generating a large amount of foam during fermentation, and will not affect the normal growth of Pseudomonas aeruginosa, and can ensure the consistency and stability of fermentation-produced rhamnolipids, thereby greatly improving the yield of fermentation-produced rhamnolipids. Using the Pseudomonas aeruginosa and method provided by the present invention, the yield of rhamnolipids can reach more than 100 g / L, which is much higher than the highest level reported. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1A schematic diagram of the rhamnolipid production capability of each strain screened for the present invention;

[0019] Figure 2 This is a schematic diagram of the phylogenetic tree of strain HY-8;

[0020] Figure 3 This is a schematic diagram of the growth curve of strain HY-8;

[0021] Figure 4 This is the colony morphology of strain HY-8;

[0022] Figure 5 This is the electron microscope scanning image of strain HY-8;

[0023] Figure 6 A schematic diagram of the ethanol tolerance of each strain screened in the present invention;

[0024] Figure 7 Schematic diagram of the effect of defoaming agents on the rhamnolipid production ability of strain HY-8. DETAILED DESCRIPTION

[0025] The present invention provides a new strain of Pseudomonas aeruginosa HY-8, which was deposited in Guangdong Microbiological Culture Collection Center (GDMCC) on October 24, 2024, with a deposit number of GDMCC NO: 65339. The 16S rDNA sequence of the Pseudomonas aeruginosa HY-8 is shown in SEQ ID NO: 1.

[0026] The Pseudomonas aeruginosa has a very strong ability to secrete rhamnolipid and can tolerate ethanol above 10% (v / v).

[0027] Based on the Pseudomonas aeruginosa, the present invention also provides a method for producing rhamnolipids, using the Pseudomonas aeruginosa to ferment and produce rhamnolipids. A preferred solution is: adding 2% to 10% of the volume of the fermentation liquid anhydrous ethanol during fermentation. A preferred solution is: using vegetable oil and / or glycerol as a carbon source during fermentation, for example, using any one or a mixture of rapeseed oil, palm oil, sunflower oil, and glycerol as a carbon source.

[0028] The technical solutions in the embodiments of the present invention will be described clearly and completely below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art. The data obtained are all average values ​​obtained after at least 3 repetitions, and all the data obtained in each repetition are valid data.

[0029] The culture medium and reagents used in the examples are as follows:

[0030] 1. Broth culture medium: 2.5 g sodium chloride, 2.5 g peptone, 1.25 g beef, and 0.5 g yeast powder are dissolved in water and the volume is adjusted to 250 mL. The pH is adjusted to 7.0. 5 g agar is added to the corresponding solid culture medium. Sterilize at 115°C for 30 min.

[0031] 2. Seed culture medium: peptone 20 g / L, glycerol 10 g / L, K2HPO4 1.5 g / L, MgSO4·7H2O 1.5 g / L, pH 7.0-7.2; sterilize at 115℃ for 30 min.

[0032] 3. Fermentation medium: rapeseed oil 45g / L, sodium nitrate 8g / L, yeast powder 1.0g / L, potassium dihydrogen phosphate 1.0g / L, magnesium sulfate 0.2g / L, sodium chloride 1.5g / L, calcium chloride 0.2g / L, pH 7.0-7.2; sterilize at 115℃ for 30min.

[0033] 4. NB culture medium: 3 g beef extract, 5 g peptone, 5 g NaCl, 1 L water.

[0034] 5. Select medium (hexadecyl trimethylammonium bromide-methylene blue): KHPO4 0.7 g / L, Na2HPO4 0.9 g / L, NaNO3 2 g / L, MgSO4 0.4 g / L, CaCl2 0.1 g / L, carbon source (glucose, vegetable oil, glycerol, etc.; rapeseed oil used in the example) 20 g / L, CTAB 0.2 g / L, methylene blue 0.005 g / L, yeast powder 1 g / L, agar powder 20 g / L, trace element mother solution 1 mL. Adjust pH to 7.0 and sterilize at 121°C for 20 min.

[0035] Trace element mother solution: FeCl3·6H2O 0.27g / L, ZnSO4·7H2O 2.67g / L, CuSO4·5H2O0.35g / L, MnSO4·H2O 1.68g / L, H3BO3 1.5g / L, dissolved in 1L deionized water, adjust pH=2.0, no need to sterilize, refrigerate at 4℃ for use.

[0036] 6. Enrichment medium: peptone 20 g / L, vegetable oil (rapeseed oil used in the example) 10 g / L, K2HPO4 1.5 g / L, MgSO4·7H2O 1.5 g / L, pH adjusted to 7.0-7.2.

[0037] Example 1: Screening and identification of microorganisms

[0038] 1. Screening

[0039] Sampling (soil) was taken from the wastewater outlet of the brewery. 1 g of soil sample was weighed and placed in enrichment medium. The soil sample was cultured in a shaker at 30 °C for 24 h. The supernatant was then diluted (10 -1 ,10 -2 ,10 -3 ,10 -4 ,10 -5 ), 100 μL of each was spread on the selection medium (three parallels were set for each gradient), and cultured in a 37°C constant temperature incubator for 2 to 4 days.

[0040] Pick the blue-green colonies that grow well on the selection medium (plate), streak them on the new selection medium, place them in a 37°C constant temperature incubator for 2 days, and then streak them again. This process is repeated for three generations to obtain pure strains. Five strains with the ability to produce rhamnolipids were screened, namely HQ-1, HQ-2, HY-6, HY-7 and HY-8.

[0041] HQ-1, HQ-2, HY-6, HY-7 and HY-8 were inoculated into the fermentation medium at an inoculum of 6% and cultured in a shaking incubator at 32°C and 240 rpm for 120 h. The rhamnolipid production of each strain was determined by the anthrone-sulfuric acid method. Each group was repeated 3 times and the results were averaged. Figure 1 As shown. The results showed that the yield of strain HY-8 was much higher than that of other strains, reaching 99.8 g / L. In addition, strain HY-8 could produce 32.5 g / L and 43.2 g / L of rhamnolipids at pH 5 and pH 8 (both at 30°C); at 20°C and 40°C (both at pH 6), the rhamnolipid yields were 25.6 g / L and 39.6 g / L, respectively. It should be noted that small-scale shaking culture in the laboratory (rather than expanded culture with continuous ventilation and stirring in the fermentation tank) will not produce a large amount of foam that will have a significant impact on the fermentation process.

[0042] 2. Identification

[0043] Extract the genomic DNA of the target strain, and clone 16S rDNA from the genomic DNA with universal primers for bacterial 16S rDNA (27F: AGA GTT TGA TCCTGG CTC AG; 1492R: TAC GGT TAC CTT GTT ACG ACT T). Send the PCR results to Shanghai Sangon Biotech Co., Ltd. for sequencing, and perform BLAST comparison on the Gene Bank website (http: / / www.nicbi.nlm.nih.gov / ) to find and download the gene sequence of the standard strain, and use MEGA7.0 software to calculate the genetic distance between different strains and draw a phylogenetic tree. The 16S rDNA sequencing results of strain HY-8 are shown in SEQ ID NO 1.

[0044] The target strain was compared with the 16S rDNA of the Bacillus model strain, and the phylogenetic tree was constructed using MEGA7.0, as shown in Figure 2 As shown, the genetic distance between strain HY-8 and Pseudomonas aeruginosa is the closest; the sequence similarity between strain HY-8 and Pseudomonas aeruginosa is 99%.

[0045] The growth curve of strain HY-8 was shown in the fermentation medium at 32°C. Figure 3 After 72 hours of culture, the colony morphology is as shown. Figure 4 As shown, the electron microscope scanning image of the microorganism is as follows Figure 5 HY-8 is a Gram-negative bacterium, with flat and moist colonies, gray-green in color, and can tolerate 10% ethanol. The suitable growth conditions are: 30°C, pH 6, weakly acidic environment.

[0046] Based on the above results, strain HY-8 was identified as Pseudomonas aeruginosa; it was deposited in Guangdong Microbiological Culture Collection Center (GDMCC) on October 24, 2024, with the deposit number: GDMCC NO: 65339. Strains HQ-1, HQ-2, HY-6, HY-7 and HY-8 were all identified as Pseudomonas aeruginosa.

[0047] Example 2: Demonstration of strain tolerance to ethanol

[0048] The strains HQ-1, HY-6 and HY-8 selected in Example 1 were inoculated into the seed culture medium respectively and cultured with shaking at 37°C overnight to make the bacteria in the logarithmic growth phase. The OD 600 The seed solution of each strain was obtained.

[0049] 1 mL of each seed solution was inoculated into 100 mL of NB medium, and anhydrous ethanol was added to the NB medium to adjust the ethanol concentration in the NB medium to 1%, 2.5%, 5%, 7.5% and 10%, respectively, so that strains HQ-1, HY-6 and HY-8 could survive in different ethanol concentration environments, with 3 replicates in each group. Each group was cultured at 37°C for 24 h, and the OD of each group was measured. 600 , the result is averaged, the result is Figure 6 shown.

[0050] The results showed that as the concentration of ethanol increased, the OD of Pseudomonas aeruginosa in each group 600 All of them continued to decline. Among them, ethanol had the most obvious inhibitory effect on the growth of HQ-1. When the ethanol concentration was 10%, HQ-1 and HY-6 could not survive at all, while HQ-8 still maintained good survival vitality.

[0051] Example 3: Demonstration of the ability of strain HY-8 to produce rhamnolipids by fermentation

[0052] Choose Bailun five-tank fermenter, the basic settings are: dissolved oxygen 60%, ventilation 4L / min, 20-400rpm dissolved oxygen associated stirring.

[0053] The strain HY-8 was cultured in a shake flask to obtain seed solution. The OD of the seed solution was 600 =1. Transfer the sterilized fermentation medium into the fermenter, and then access the seed liquid under a sterile environment. Set up 3 treatments: Experimental group: Use a foam circulation device (connect a tube to the exhaust end of the fermenter to introduce a large amount of foam produced by fermentation into the foam collection tank, and then use a peristaltic pump to pump the liquid formed by the foam in the foam collection tank back to the fermenter), add anhydrous ethanol to the foam collection tank, each time the amount is 20mL, starting from the 36th hour, once every 24 hours; the ethanol concentration in the control system is 2%. Positive control group: Use a silicone oil type defoamer (dimethyl silicone oil defoamer, purchased from Hefei Xinwancheng Environmental Protection Technology Co., Ltd.) instead of anhydrous ethanol, each time the amount is 20mL, and the rest is the same as the experimental group. Blank control group: No defoamer is used, and the rest is the same as the experimental group. Samples were taken at the 24th, 48th, 72nd and 96th hours of fermentation, and the rhamnolipid content in the fermentation system was determined by the anthrone-sulfuric acid method. Three replicates were set for each treatment, and the results were averaged. The results are as follows Figure 7 shown.

[0054] The results showed that in the blank control group without defoaming agent, a large amount of foam was observed during fermentation from the 18th hour of fermentation, and the foam continued to bubble up the tank, affecting the continuity of fermentation, resulting in poor overall growth of the strain and low rhamnolipid production. Due to severe bubbling, the rhamnolipid production of the blank control group was only 54g / L at 96h of fermentation, but this is already the leading level of rhamnolipid production in existing studies. After adding the organosilicon defoamer, no significant reduction in foam was observed, and the increase in rhamnolipid production was not obvious. This may be because organosilicon cannot effectively defoam, and a large amount of nutrients are adsorbed on the surface of the foam, which reduces the nutrients in the fermentation liquid and reduces the rhamnolipid production.

[0055] Since a large amount of oxygen is required in the fermentation process of rhamnolipid production, which results in uneven formation of uniform adsorption displacement and asymmetric water film, short-chain alcohols can be used as deoxidizers, and ethanol can reduce the transfer rate of oxygen, separating oxygen from foam in a short time, thereby achieving the purpose of rapid defoaming. Adding ethanol as a defoamer can effectively control the foam, thereby achieving continuous production of rhamnolipid, with a final yield of 103g / L.

[0056] The embodiments described above are only descriptions of the preferred modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations, modifications, and substitutions made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A strain of Pseudomonas aeruginosa HY-8, characterized in that: It was deposited in Guangdong Provincial Microbiological Culture Collection Center on October 24, 2024, with the deposit number: GDMCC NO: 65339.

2. A strain of Pseudomonas aeruginosa, characterized in that: The 16S rDNA sequence of Pseudomonas aeruginosa is shown in SEQ ID NO:

1.

3. A bacterial preparation prepared using the Pseudomonas aeruginosa according to claim 1 or 2.

4. A bacterial preparation comprising the Pseudomonas aeruginosa according to claim 1 or 2.

5. A method for preparing rhamnolipid using the Pseudomonas aeruginosa according to claim 1 or 2 or the bacterial preparation according to claim 3 or 4.

6. The method according to claim 5, characterized in that: The method comprises: utilizing the Pseudomonas aeruginosa to ferment and produce rhamnolipid.

7. The method according to claim 6, characterized in that: Add ethanol to the fermentation system.

8. The method according to claim 6, characterized in that: During fermentation, vegetable oil and / or glycerol are used as carbon sources.

9. The method according to claim 6, characterized in that: The fermentation temperature is 28-35℃.

10. The method according to claim 6, characterized in that: The fermentation pH is 6-8.

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