What are the key steps in the operation of fermentation tanks?

Fermentation Tank Operation Technology

1 Technical Preparation

Before using the equipment, check whether the power supply is normal, and whether the air compressor, microcomputer control system, and circulating water system can work normally. Check whether the valves, joints, and fastening screws on the system are tightened. Start the air compressor, and use a pressure of 0.15Mpa to check whether the sealing performance of the seed tank, fermentation tank, filter, pipeline, and valve is good. The temperature gauge, speed control motor, and capacitive vortex flowmeter should be checked and calibrated according to the user manual.

2 Air Sterilization

Before feeding, the air path, material path, seed tank, fermentation tank, and defoaming tank must be sterilized with steam to eliminate all dead-end bacteria and ensure the system is in a sterile state.

2.1 Air Pipeline Sterilization

(1) The air pipeline has a three-stage pre-filter, a refrigerated dryer, and a sterilizing filter. The pre-filter and refrigerated dryer cannot be sterilized with steam, so before steam passes through the air pipeline, the valve leading to the pre-filter must be closed, allowing steam to pass through the pressure reducing valve and steam filter, then into the sterilizing filter.

(2) The filter element of the sterilizing filter cannot withstand high temperature and high pressure, so the steam pressure reducing valve must be adjusted to 0.13Mpa and must not exceed 0.15MPa.

(3) During the air sterilization process, the exhaust valve at the lower end of the sterilizing filter should be slightly opened to discharge condensate.

(4) The air sterilization time should last about 40 minutes. When the equipment is used for the first time or started after a long period of inactivity, it is best to use intermittent air sterilization, that is, after the first air sterilization, wait 3-5 hours before sterilizing again to eliminate spores.

(5) After air sterilization, the filter should be dried with air for about 20-30 minutes, and then the air path valve should be closed.

2.2 Seed Tank, Fermentation Tank, Alkali Tank, and Defoaming Tank Sterilization

(1) The seed tank, fermentation tank, alkali tank, and defoaming tank are sterilized by directly introducing steam into the tank.

(2) During sterilization, the inoculation port, exhaust valve, and material path valve on the tank should be slightly opened to allow steam to escape through these valves, while maintaining the tank pressure at 0.13-0.15Mpa.

(3) The sterilization time is 30-40 minutes; in special cases, intermittent sterilization can be used.

(4) Before sterilizing the seed tank, fermentation tank, defoaming tank, and alkali tank, the water in the jacket should be drained. After sterilization, the condensate in the tank should be drained, and the exhaust valve should be opened to prevent negative pressure in the tank after cooling, which could damage the equipment. During sterilization, the dissolved oxygen and pH electrodes should be removed to extend their service life.

3 Actual Sterilization

Actual sterilization is the process of sterilizing the culture medium with steam after it is added to the tank. The operation process for the seed tank, alkali tank, defoaming tank, and fermentation tank is the same. After air sterilization, the prepared culture medium should be added to the tank from the feeding port as soon as possible, and there should be no cooling water in the jacket at this time. The culture medium should be gelatinized before entering the tank, and the formula amount of the culture medium is generally calculated at about 70% of the total volume of the tank (for foamy culture media, about 65%, and for less foamy culture media, it can reach 75-80%). Considering the factors of condensate and inoculation amount, the amount of water added is about 50% of the total volume of the tank. The amount of water added is related to the temperature of the culture medium and steam pressure, and needs to be explored in practice. First, turn on the mechanical stirring device to mix the materials in the tank evenly at a speed of 50-100rpm. Open the steam valve and exhaust valve in the jacket to preheat the culture medium in the tank. When the temperature in the tank reaches 90℃, close the steam inlet valve of the jacket and open all steam inlet valves in the tank to introduce steam. When the tank temperature rises to 105℃, slowly open the exhaust valve to discharge the cold air from the top of the tank, and after five minutes, close the exhaust valve. When the tank pressure rises to 0.12Mpa and the temperature reaches 121-123℃, control the opening of the steam valve to maintain the tank pressure constant, and stop supplying steam after 30 minutes. Open the inlet and outlet valves of the cooling water, and introduce water into the jacket for cooling. When the pressure in the tank drops to 0.05Mpa, slightly open the exhaust valve and inlet valve to ventilate and stir, accelerating the cooling speed, and maintain the tank pressure at 0.05Mpa until the tank temperature drops to the inoculation temperature.

4 Inoculation

Inoculation is performed using flame sealing. Before inoculation, prepare alcohol cotton, forceps, tweezers, and inoculation loops in advance. The strain is placed in a triangular flask, and the inoculation amount is determined according to the process requirements. Ignite the alcohol cotton surrounding the inoculation port, use forceps or an iron rod to open the inoculation port, and at this time, air should be introduced into the tank to allow air to escape from the inoculation port. Pour the strain from the triangular flask into the tank in the middle of the flame loop. After sterilizing the inoculation port over the flame, tighten it. After inoculation, ventilation cultivation can begin, maintaining the tank pressure at 0.05Mpa.