How to operate a 200-liter liquid spawn fermenter?

How to operate a 200-liter liquid spawn fermenter?

1. Add water
Fill to a level just above the upper sight glass.

2. Add materials
Supply cold compressed air (to agitate the mixture and prevent settling), add the raw materials and an antifoaming agent (20–25 ml per 100 liters), and securely close the lid.

3. Sterilization (Boiling)
Set the vessel temperature to 125°C and the duration to 80 minutes. Turn on the "Heating" switch and verify the following: a. The exhaust valve at the top of the vessel is open; b. The water inlet and outlet valves are open; c. The temperature display is on. The vessel may make some noise during the heating process; turn off the cold air supply once the temperature reaches approximately 90–95°C. When the temperature hits 100°C, adjust the top exhaust valve to a slightly open position. Once the temperature reaches 125°C and the pressure hits 0.15 MPa, begin the sterilization timer.

4. Connect water inlet and outlet lines before sterilization ends
When the sterilization timer finishes, turn off the "Heating" switch, then open the water supply valve to begin cooling with cold water. As the vessel cools, the pressure will drop; once the pressure gauge reads below 0.08 MPa, supply cold air to the vessel. When the vessel reaches the desired temperature, close all water inlet and outlet valves; the vessel is now ready for inoculation.

5. Inoculation
Before inoculating, prepare the following: a spray bottle with 75% alcohol, the shake flask containing the inoculum, a flame ring, a lighter, a wet towel, and a stainless steel rod. Sterilize the area around the inoculation port using 75% alcohol and remove the aluminum foil covering the shake flask. Soak the flame ring in 95% alcohol, place it around the inoculation port, and reduce the tank pressure to 0.01 MPa. Ignite the flame ring, use the metal rod to unscrew the inoculation port cap (stopping just before the final turn), and—simultaneously opening the cap and closing the exhaust valve—bring the shake flask over. Pass the mouth of the shake flask through the flame to sterilize it, remove the cotton plug, and pour in the seed culture. Briefly flame the inoculation port cap, quickly place it back on, and tighten it securely with the metal rod. Extinguish the flame with a wet towel to complete the inoculation process. Finally, adjust the aeration/pressure to approximately 0.025–0.05 MPa for cultivation and use a marker to label the tank with the inoculation time, date, and strain name.

6. Cultivation
Once inoculation is complete, set the cultivation temperature to approximately 25°C (note that the temperature may vary depending on the strain) and turn on the "thermal insulation" switch. Adjust the aeration and pressure to the required levels and allow the pressure to stabilize.