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Fermentation method with mechanical stirring fermenter

Principle
Start with test-tube strains, increase the culture by scale-up, and finally ferment Bacillus subtilis using a mechanically stirred tank. Shaking flask culture is a commonly used ventilation culture method in the laboratory. The triangular flask containing liquid culture is placed on a shaker for shaking culture to meet the oxygen demand for the growth, reproduction and production of many metabolites of microorganisms. It is a laboratory exploration. Common process conditions
1. Experimental materials
Bacillus subtilis, seed medium, fermentation medium, constant temperature shaker, sterile Pasteur pipette, test tube, conical flask, fermenter.
 2. Experimental method
1. Process flow
Test tube slanted bacteria (12 h, 37 °C) → inoculation (1 ring of fungus) → shaking flask seed culture solution, 250 mL conical flask shaking culture (12 h, 37 °C) → seed culture solution, inoculation (1%- 5%) → fermentation in a mechanically stirred fermenter.
2. Preparation of Fermentor Seed Liquid
Shake flask seed medium: 20 g glucose, 1 g beef extract, 5 g peptone, 5 g NaCl, 1 000 ml H2O, natural pH, autoclave at 121 °C for 20 min.
Shake flask seed medium is divided into 250ml Erlenmeyer flasks, 50ml per flask. Pick a ring of bacteria from the inclined surface, insert it into the seed medium of the shaker flask, and shake it for 12 hours at 37° C. and 150 rpm, which is the seed liquid.
3. Preparation of Fermentation Medium
Fermentation medium: sucrose 20 g, starch 20 g, yeast extract 1 g, peptone 5 g, NaNO3 3 g, KH2PO4 5 g, CaCO3 10 g, H2O 1 000 ml; pH 7.0.
Starch gelatinization: starch: water = 1:4, add water preheated to 60°C, stir while adding, and boil for 10 minutes to form a paste (keep stirring during the boiling process).
 4. Fermentation
①Empty elimination: sterilization of empty tanks.
②Actual elimination: Pour 5L of fermentation medium from the injection port into a 15L fermenter, and close the lid. After checking that the fermenter is well installed, sterilize at 105°C for 15 minutes.
③Inoculation: The flame inoculation method can be used, and an alcohol circle is placed at the inoculation port. After ignition, the bacteria are quickly poured into the fermenter through the flame circle for timed cultivation.
④Fermentation: After the seeds are cultivated, connect the seeds to the fermentation tank, the inoculation amount is 1-5%, 36°C-37°C temperature-controlled ventilation cultivation, stirring speed 200 rpm, 0-1 h, ventilation ratio 1 : 0.5 to 0.8; after 35 hr, adjust the speed to 380 rpm; 24 to 28 hours, ventilation 1:1.0. Vitality was measured every 6 hours, appearance sugar was measured once; pH was measured every 2 hours, and ventilation volume, rotational speed, temperature, etc. were recorded. When monitoring the fermentation process, pay attention to observe the structure of the fermenter, record the measurement results in the table, and conduct microscopic inspection at the same time. The bacterial concentration was measured regularly, and the fermentation was terminated when the bacterial concentration reached the highest.
⑤ Sampling: From the beginning of the culture operation, take a sample every 6 hours. Before and after sampling, flush the sampling port with steam.
 
Precautions
1. Before the test tube is connected to the shake flask, the shake flask medium should be set as a blank control for streaking the broth plate, and check whether the shake flask seed medium is completely sterilized.
2. Before the seed culture liquid is connected to the fermenter, the microscopic examination of the seed culture liquid should be carried out to check whether there is any contamination of bacteria.