A strain of Pseudomonas aeruginosa co-producing rhamnolipid and lipopeptide
A Pseudomonas aeruginosa, rhamnolipid technology, applied in bacteria, microorganism-based methods, biochemical equipment and methods, etc., can solve problems such as low yield, and achieve high yield, good feasibility, and excellent application performance. Effect
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Embodiment 1
[0018] Embodiment 1: Separation, screening and identification of strains
[0019] (1) Enrichment screening of strains
[0020] The Pseudomonas aeruginosa S1 of the present invention comes from soil polluted by crude oil, and the strain can grow on an LB plate at 37° C., and the colonies are green and the surface is wet. The Pseudomonas aeruginosa is a rhamnolipid and lipopeptide co-production strain screened from soil polluted by crude oil.
[0021] Take 10g of soil contaminated by crude oil, add 40ml of sterile water, and incubate on a constant temperature shaker at 37°C for 15 minutes. Then take 2mL supernatant and put it into 60mL selection medium for enrichment culture for 24h. The composition of the selection medium is: 3% (v / v) corn oil, 4g / L NaNO 3 , 0.2g / L MgSO 4 , 1.0g / L NaCl, 1.0g / L KCl, 0.04g / L CaCl 2 , 5ml / L 85%H 3 PO 4 ;pH=7.0. After culturing for 24 hours, the culture solution was serially diluted, applied to the screening solid medium, and cultured in an...
Embodiment 2
[0028] Embodiment 2: Determination of fermentation capacity
[0029] (1) Seed culture: Resuscitate Pseudomonas aeruginosa S1 glycerol cryopreservation tubes stored at -80°C in warm water at 40-60°C, inoculate 1.4 mL into 60 ml seed medium, and culture in a shaker at 37°C and 260 rpm for 2 days. The described seed medium consists of: 4g / L NaNO 3 ; 1.0g / L NaCl; 1.0g / L KCl 1.0; 0.2g / L MgSO 4 ;0.04g / LCaCl 2 ; 5mL / L H 3 PO 4 3% (v / v) corn oil; pH 6.6.
[0030] (2) Fermentation culture: the seed culture medium that cultivates 2 days is inoculated to fermentation culture medium by the inoculum size of 3% v / v, at 37 ℃, under the condition of 260rpm, carry out by a definite date 6 days fed-batch (fed-batch ) fermentation, in order to ensure sufficient carbon source in the fermentation process, 1.5% corn oil was added on the 3rd and 4th days respectively. Fermentation medium composition: 4-12g / L NaNO 3 ; 1.0g / L NaCl; 1.0g / L KCl; 0.2g / L MgSO 4 ;0.04g / L CaCl 2 ; 5mL / L H 3 PO 4 ...
Embodiment 3
[0035] Embodiment 3: fermentation performance optimization
[0036] (1) Seed culture: Resuscitate Pseudomonas aeruginosa S1 glycerol cryopreservation tubes stored at -80°C in warm water at 40-60°C, inoculate 1.4 mL into 60 ml seed medium, and culture in a shaker at 37°C and 260 rpm for 2 days. The liquid volume of the shake bottle is 60mL / 250mL. The described seed medium consists of: 4g / L NaNO 3 ; 1.0g / L NaCl; 1.0g / L KCl 1.0; 0.2g / L MgSO 4 ;0.04g / L CaCl 2 ; 5mL / L H 3 PO 4 3% (v / v) corn oil; pH 6.6.
[0037] (2) Fermentation culture: inoculate the seed medium cultivated for 2 days into the fermentation medium at an inoculum size of 3% v / v, and carry out fed-batch (fed-batch) for 5 days at 37°C and 260rpm For fermentation, 1.5% (v / v) corn oil was added on the 2nd and 3rd day. Fermentation medium composition: 12g / L NaNO 3 ; 1.0g / L NaCl; 1.0g / L KCl; 0.2g / L MgSO 4 ;0.04g / LCaCl 2 ; 5mL / L H 3 PO 4 ; 3-10% (v / v) corn oil; 0.5mg / L FeSO 4 ·7H 2 O; 0.1-2mg / L FeCl 3 ·6H 2 ...
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