Laboratory stainless steel fermentation tankAs a core equipment for microbial cultivation, metabolite synthesis, and biological reaction research, it is widely used in microbiology, biotechnology, food science, and other fields due to its advantages of corrosion resistance, easy cleaning, and precise temperature control. The operational standardization directly determines the stability and experimental repeatability of the fermentation system, while the sterilization process is a key prerequisite for avoiding bacterial contamination and ensuring experimental success. This article will provide a detailed overview of the standard operating procedures and core sterilization methods for stainless steel fermentation tanks in the laboratory, offering practical guidance for laboratory personnel.
1、 Laboratory stainless steel fermentation tank operation process
The operation of stainless steel fermentation tanks in the laboratory needs to follow a closed-loop logic of "preparation debugging fermentation ending", with the core being precise temperature control pH、 Key parameters such as dissolved oxygen and stirring meet the growth and metabolic needs of microorganisms.
（1） Preparation before operation: Build a solid experimental foundation
Equipment inspection: Check the status of the core components of the fermentation tank one by one - the tank body and seals are not damaged or leaking; The stirring blade rotates flexibly without jamming; The temperature sensor, pH electrode, dissolved oxygen electrode and other probes are clean and free of scratches; The intake pipe, feeding pipe, exhaust pipe and other pipelines are unobstructed and unobstructed; Safety components such as safety valves and pressure gauges are within their valid calibration period. At the same time, check whether the supporting equipment (such as air compressor, chiller, peristaltic pump, sterilization pot) is operating normally.
Material preparation: Accurately prepare the culture medium according to the fermentation formula, ensuring that each component (carbon source, nitrogen source, inorganic salts, trace elements, etc.) is weighed accurately and fully dissolved to avoid clumping. For easily oxidizable or thermosensitive components, they need to be separately prepared and supplemented using sterile filtration (0.22 μ m filter membrane). Prepare the bacterial suspension to ensure that the bacterial activity meets the standard and the concentration meets the inoculation requirements.
Pipeline connection and sealing: Connect each pipeline according to the fermentation process - the intake pipe is connected to the sterile air system, the exhaust pipe is connected to the condensation device and exhaust gas treatment system, the replenishment pipe is connected to the sterile replenishment bottle, and the sampling pipe is equipped with a sterile sampling valve. After connection, tighten the nuts of each interface to ensure a tight seal (which can be verified by subsequent pressure testing), avoiding air leakage or bacterial invasion during the fermentation process.
（2） Equipment debugging: precise parameter setting
No load debugging: Connect the main power supply of the fermentation tank, start the control system, and sequentially test the stirring system (adjust the speed to 50-100rpm, observe the smoothness of the stirring blade operation), temperature control system (set 37 ℃, check whether the heating or cooling module starts and stops normally), pH control system (calibrate the pH electrode with standard buffer solution to ensure reading error ≤ ± 0.05), and dissolved oxygen control system (calibrate the dissolved oxygen electrode, observe whether the reading change is sensitive after introducing air).
Media loading and parameter presetting: The prepared media is injected into the fermentation tank through a sterile funnel, and the filling volume is controlled at 50% -70% of the effective volume of the tank (to avoid foam overflow or insufficient dissolved oxygen during fermentation). After closing the feeding port, preset the core parameters in the control system - temperature (such as bacteria 37 ℃, fungi 28 ℃), stirring speed (set 50-300rpm according to the strain requirements), ventilation rate (usually 0.5-2vvm, that is, 0.5-2 volumes of sterile air per hour per volume of culture medium), pH value (such as bacteria 7.0-7.2, yeast 4.5-5.5), and feeding strategy (feeding time, feeding rate).
（3） Fermentation operation: dynamic monitoring and regulation
Vaccination operation: Aseptic operation technology is used for inoculation - the inoculation port is wiped with alcohol for disinfection. After opening the inoculation port, the bacterial suspension is quickly injected into the tank, and the inoculation port nut is immediately tightened after injection. The vaccination process should be fast and standardized to avoid prolonged exposure to air and the introduction of miscellaneous bacteria.
Parameter monitoring and adjustment: Record temperature every hour during the initial fermentation period pH、 Dissolved oxygen, stirring speed and other parameters can be recorded once every 2-4 hours after stabilization. Dynamic regulation based on parameter changes: When the temperature deviates from the set value, it is adjusted through heating or cooling modules; Add sterile ammonia water or NaOH solution when pH drops, and add sterile hydrochloric acid or organic acid when pH rises; When the dissolved oxygen is too low, the stirring speed can be increased, the ventilation rate can be increased, or pure oxygen can be introduced; When a large amount of foam appears, manually add sterile defoamer or start the automatic defoamer system.
Sampling and testing: Samples should be taken at regular intervals according to experimental requirements. Before sampling, the sampling valve should be disinfected with alcohol and a small amount of fermentation broth should be drained to rinse the pipeline before collecting the samples. The sample needs to be promptly tested for bacterial concentration (such as OD600 measurement), metabolite content (such as high-performance liquid chromatography analysis), residual components of the culture medium, etc., to provide a basis for judging the fermentation process and adjusting parameters.
（4） Fermentation Closing: Standardized Shutdown and Cleaning
Shutdown operation: When the fermentation reaches the preset endpoint (such as peak bacterial concentration and maximum production of metabolites), first turn off the heating/cooling module, stirring system, and ventilation system, and then cut off the main power supply. Slowly open the exhaust valve to relieve pressure, and after the pressure inside the tank drops to atmospheric pressure, open the discharge port to collect the fermentation broth.
Preliminary cleaning: Timely empty the residual fermentation liquid in the tank, rinse the inside of the tank, stirring blade and probe surface with tap water, and remove obvious bacterial adhesion and culture medium residue. For pipeline systems, high-pressure tap water can be introduced for flushing to ensure no residual fluid accumulation.
2、 Sterilization process of laboratory stainless steel fermentation tank
Bacterial contamination is one of the main reasons for the failure of fermentation experiments. The sterilization of stainless steel fermentation tanks in the laboratory should follow the principle of "comprehensive coverage and thorough killing", with the core focus on sterilization of the tank body, culture medium, pipelines, and accessories. The commonly used method is wet heat sterilization (high-pressure steam sterilization), and special scenarios can be combined with dry heat sterilization or chemical sterilization.
（1） Preparation before sterilization: investigation and pretreatment
Equipment and pipeline inspection: Confirm that the sealing components of the fermentation tank (such as silicone sealing rings) are not aging and that the pipeline interfaces are not loose; Check if the water level of the sterilization pot meets the standard, and if the safety valve and exhaust valve are functioning properly. For components that are not resistant to high temperatures (such as some pH electrodes and dissolved oxygen electrodes), they need to be removed in advance and replaced with sterilization specific plugs or treated with separate aseptic methods.
Tank and pipeline pretreatment: Rinse the tank and pipeline with deionized water to remove residual detergent or impurities. After injecting the culture medium into the tank, turn on the stirring blade and stir for 5-10 minutes to ensure even distribution of the culture medium and avoid local clumping affecting the sterilization effect.
（2） Core sterilization method: Wet heat sterilization operation (commonly used)
Wet heat sterilization utilizes the high temperature (121 ℃) and high humidity of high-pressure steam to kill microorganisms and spores. It is suitable for high-temperature resistant components such as tanks, culture media, and pipelines. The specific process is as follows:
Sealing and air intake: Close all openings of the fermentation tank (feeding port, sampling port, discharge port), tighten the nuts of each interface to ensure the tank is sealed. Connect the exhaust and intake pipes of the fermentation tank to the sterilization pot or specialized sterilization steam generator, open the intake valve, slowly introduce steam, and eliminate the cold air in the tank and pipeline (residual cold air can cause local temperature deficiency and incomplete sterilization).
Boosting and insulation: After the exhaust pipe continuously discharges uniform steam (without cold air inclusions), close the exhaust valve and start boosting. When the pressure inside the tank rises to 0.1 MPa (gauge pressure) and the temperature reaches 121 ℃, maintain this pressure and temperature for 30-60 minutes (adjusted according to the composition of the culture medium: extended to 60 minutes for difficult to sterilize components such as starch and protein, and 30 minutes for ordinary culture medium). During the sterilization process, closely monitor the pressure and temperature. If the pressure drops, steam should be replenished in a timely manner.
Pressure reduction and cooling: After sterilization, slowly open the exhaust valve to relieve pressure. When the pressure drops to 0.02 MPa, turn on the cooling system (such as a chiller) to cool down the tank. When the temperature inside the tank drops to 40-50 ℃ (to avoid bacterial inactivation caused by high temperature inoculation and medium solidification caused by low temperature inoculation), and the pressure drops to atmospheric pressure, turn off the cooling system and prepare for inoculation.
（3） Auxiliary sterilization method: suitable for special scenarios
Dry heat sterilization: For components that cannot be sterilized by wet heat sterilization (such as metal sampling spoons and accessories inside sterile workstations), place them in a dry heat sterilization box and sterilize them at 160-180 ℃ for 2-3 hours. After sterilization, cool them naturally to room temperature for later use.
Chemical sterilization: For pipelines or surfaces that are not resistant to high temperatures (such as some plastic pipelines and sensor probes), wipe and disinfect them with 75% alcohol or soak them for 30 minutes, then rinse them with sterile physiological saline to remove residual alcohol and avoid chemical reagents inhibiting bacterial strains.
Sterilization of sterile air system: The air introduced during the fermentation process needs to be sterile and can be treated by high-temperature sterilization (heating the air to 121 ℃ for 30 minutes) or filter cartridge filtration (using a 0.22 μ m sterile filter cartridge) to ensure that the air introduced into the tank is free of bacteria.
（4） Verification and maintenance after sterilization
Sterilization effect verification: The "blank culture method" can be used for verification - without inoculation after sterilization, the fermentation tank is placed at the fermentation set temperature and cultured for 24-48 hours. If the culture medium is not turbid and there is no bacterial growth, it indicates that the sterilization is qualified; Biological indicators (such as thermophilic Bacillus spores) can also be used, and the indicator can be cultured after sterilization. If the indicator does not change color, sterilization is qualified.
Component reset and storage: After sterilization is qualified, the probes (such as pH and dissolved oxygen electrodes) that were removed in advance will be rinsed with sterile physiological saline and installed for reset to ensure interface sealing. Fermentation tanks that are not immediately used should be kept dry by blowing sterile air inside and closing all openings for later use.
3、 Key precautions for operation and sterilization
Safety priority: It is strictly prohibited to open the tank or sterilization pot without authorization during the sterilization process. When unloading pressure, it is necessary to operate slowly to avoid sudden drops in pressure that may cause the culture medium to boil; When operating the mixing system, it is forbidden to insert hands or tools into the tank to prevent mechanical damage.
Aseptic operation runs through the entire process: inoculation, sampling, feeding and other processes need to be carried out in a sterile operating table. Operators need to wear sterile clothing, gloves, and masks in a standardized manner to avoid introducing miscellaneous bacteria artificially.
Regular maintenance of equipment: Check the aging condition of the sealing components of the fermentation tank every month and replace them in a timely manner; Calibrate temperature sensors, pH electrodes, dissolved oxygen electrodes, and pressure gauges every 3-6 months to ensure accurate parameter detection; Clean the tank in a timely manner after sterilization to prevent residual culture medium from corroding the stainless steel inner wall.
Complete parameter recording: Detailed record of temperature during operation pH、 Parameters such as dissolved oxygen and stirring speed, as well as information on sterilization pressure, temperature, and holding time, provide a basis for experimental repetition and problem investigation.
In conclusion,Laboratory stainless steel fermentation tankThe operation and sterilization process should follow the core principles of "precise control and sterile assurance". Standardized operating procedures can ensure stable fermentation parameters and improve experimental reproducibility; A strict sterilization process can prevent bacterial contamination from the source and ensure the success of experiments. By proficiently mastering the above process and precautions, the experimental value of fermentation tanks can be fully utilized, providing reliable support for microbial research and bioengineering experiments.