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5. Sterilization of Components

Items: Filter element (with shell or as a whole), filter element cover, air inlet pipe, inoculation gun, and hose.

Steps: Wrap pipes/hoses with 8-10 layers of medical degreased gauze; position filter elements downward, ensure no pipe folds, and cover openings with polypropylene film. Exhaust at 0.05 MPa (2 cycles) or for 5-10 minutes. Sterilize at 121℃ for 60 minutes.

6. Loading

Close lower air inlet and inoculation valves. Pour nutrient solution into the fermentation tank via the feed port, add 15ml defoamer, and rinse the cooking pot.

Loading volume: 60%-80% of tank capacity (e.g., 45±5L for a 70L tank), with height 10cm above the sight glass upper edge. Tighten the feed port cover to prevent air leakage.

7. Culture Medium Sterilization

Close all valves, power on. When pressure reaches 0.05MPa, exhaust to zero twice (second time for 1 minute).

Maintain 121-125℃ (0.12-0.14MPa) for 60 minutes, then close the sterilization switch.

During sterilization, open the discharge port 3 times to release 3-5L of material and sterilize valve pipelines.

8. Culture Medium Cooling

Goal: Cool to 25-28℃.

Method 1: Use circulating cooling water. When pressure drops to 0.05-0.08MPa, turn on the air pump, adjust tank pressure to 0.02MPa, and prepare for inoculation when temperature <28℃.

Method 2: Ventilation. After final discharge, flame the air inlet (10-20s) and connect the pipe. Adjust pressure (tank pressure < gas storage tank pressure by 0.02MPa) to allow air flow, cooling via gas agitation. Stop cooling water when temperature is 2℃ above the culture temperature.

A 70L tank takes ~60 minutes to cool.

9. In-Tank Inoculation

Preparation: Flame ring (alcohol-soaked cotton), lighter, 95% alcohol, heat-resistant gloves.

Steps: Reduce tank pressure to near 0, ignite the flame ring over the feed port, open the cover, flame-sterilize the shake flask mouth, add 1000-1500ml of shake flask liquid, re-cover and flame-sterilize the port, tighten, and extinguish the flame.

Restore micro-pressure (0.01-0.02MPa), set culture temperature, and check for backflow.

10. Cultivation

Start the control cabinet; parameters: 22℃, pH 6.5, 84-96h duration, tank pressure 0.02-0.04MPa, ventilation 1:0.8 (air volume = 80% of culture volume/min).

Avoid power/air compressor shutdown. If power fails, close valves immediately to maintain 0.02-0.04MPa; use backup power if pressure drops.