Bacillus subtilis fermentation culture experiment

Summary

Starting with test tube strains, the culture is added by expanding step by step, eventually fermenting Bacillus subtilis using a mechanical mixing tank. Shaking flask culture is a commonly used ventilated culture method in laboratories, whereby triangular flasks containing liquid cultures are placed on a shaker and oscillated to meet the oxygen requirements of microorganisms for growth, reproduction and the production of many metabolites, and it is a common method used in laboratories for figuring out the conditions of the process.

Operation method

Mechanical stirred fermenter fermentation method

Principle

Starting with test tube strains, the culture is added by expanding step by step, eventually fermenting Bacillus subtilis using a mechanical mixing tank. Shaking flask culture is a commonly used ventilated culture method in laboratories, whereby triangular flasks containing liquid cultures are placed on a shaker and oscillated to meet the oxygen requirements of microorganisms for growth, reproduction and the production of many metabolites, and it is a common method used in laboratories for figuring out the conditions of the process.

Materials and Instruments

Bacillus subtilis
Seed culture medium Fermentation medium
Thermostatic shakers Sterile pipettes Test tubes Triangular flasks Fermenters

Move

I. Experimental materials
Bacillus subtilis, seed medium, fermentation medium, thermostatic shaker, sterile pipettes, test tubes, triangular flasks, fermenter.
II. Experimental methodology
1. ProcessesTest tube slant seed (12 h, 37 ℃) → inoculation (1 ring bacterial moss) → shaking bottle seed culture in 250 mL triangular flask oscillation culture (12 h, 37 ℃) → seed culture, inoculation (1%-5%) → mechanical stirring fermentation tank fermentation.
2. Preparation of fermenter seed liquorShake flask seed medium: glucose 20 g, beef paste 1 g, peptone 5 g, NaCl 5 g, _{H2 O} 1 000 ml, pH natural , autoclave sterilized at 121°C for 20 min.Shaking bottle seed medium was divided into 250 ml triangular bottle, 50 ml per bottle, pick a ring of strains from the slant, access to the shaking bottle seed medium, 37 ℃, 150 rpm oscillation culture for 12 hours that is the seed liquid.
3. Fermentation media preparation
Fermentation medium: 20 g of sucrose, 20 g of starch, 1 g of yeast paste, 5 g of peptone, 3 g of _NaNO3, 5 g of _KH2PO4, 10 g of _CaCO3, 1 000 ml of H2O; pH 7.0.
Starch pasting: starch: water = 1:4 added to preheated to 60 ℃ water, stirring while adding, boiling for 10 minutes into a paste (constant stirring during the boiling process).
4. Fermentation (demonstration)
①Air elimination: empty tank sterilization.
②Substantial sterilization: Pour 5 L of fermentation medium from the inlet into a 15 L fermenter and close the lid. After checking that the fermenter is well installed, sterilize at 105℃ for 15 minutes.
③ Inoculation: Flame inoculation method can be used, where an alcohol ring is placed at the inoculation port, ignited, and then the strain is rapidly poured into the fermenter through the flame ring for timed cultivation.
④Fermentation: after the seed culture is ready, the seed is received into the fermenter, inoculation amount 1-5%, 36°C-37°C controlled temperature ventilation culture, stirring speed 200 rpm, 0-1 h, ventilation ratio l: 0.5-0.8; after 35 hr, adjust the speed of 380 rpm; 24-28 hr, ventilation 1: 1.0. Measure the vitality every 6 hr, appearance of Sugar every 6 hours; every 2 hours to measure pH, record ventilation, speed, temperature and so on. When monitoring the fermentation process, pay attention to observe the structure of the fermenter, record the results in the table, and examine it by mirror at the same time. Measure the bacterial concentration regularly and end the fermentation when the bacterial concentration reaches the maximum.
⑤ Sampling: take samples every 6h from the beginning of the culture operation. Before and after sampling, flush the sampling port with steam.

Caveat

First, before the test tube is connected to the shaking bottle, the shaking bottle medium should be set up with a broth plate scribing blank control to check whether the shaking bottle seed medium is sterilized thoroughly.Second, before the seed culture solution is connected to the fermenter, the seed culture solution should be microscopically inspected to check whether there are stray bacteria contamination.

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