The seed tank in the workshop uses liquid deep culture technology to achieve efficient reproduction and activation of strains from small quantities to large quantities, from the laboratory stage to the industrial production stage, providing sufficient quantities of seeds with appropriate activity for fermentation production, ensuring the smooth progress of the fermentation process.
Seed preparation in the production workshop is usually called vegetative preparation (mycelium preparation), and vegetative culture is deep culture in seed tanks. The purpose of seed preparation in the production workshop is to grow a limited number of strains in the seed bottle and multiply them into a large number of vegetative bodies to shorten the time of fermentation tank reproduction. Seed preparation is carried out in seed tanks using liquid deep culture.
The spores or nutrient bodies (mycelium) prepared in the laboratory are transplanted to seed tanks for expanded culture. Although the culture medium of the seed tank varies with different strains, the principle is to use ingredients that are easily utilized by the bacteria, such as glucose, corn pulp, phosphates, etc. At the same time, sufficient sterile air must be supplied and stirred continuously to ensure that the nutrient bodies are evenly distributed in the culture medium and the same culture conditions are obtained.
There are several methods for seed tank inoculation. Spore suspension is generally inoculated by micropore inoculation. Seed inoculation with shake flask vegetative body can be connected to the seed tank under flame protection or by pressure difference method. The transfer method between seed tanks or fermentation tanks mainly adopts pressure difference method, and the seed is transferred through the seed transfer pipeline. During the transfer process, it is necessary to prevent the surface pressure of the receiving tank from dropping to zero, otherwise it will cause bacterial contamination.
The process of seed preparation is a step-by-step expansion process, which varies according to the product variety. It is generally divided into primary seed preparation, secondary seed preparation, and tertiary seed preparation. Why should we expand the culture step by step? Since the number of spores and shake flask mycelium prepared in the laboratory is limited, if a small amount of strains are directly connected to a fermentation tank of dozens of cubic meters, on the one hand, the strain growth time will be very long, which will prolong the non-production cycle in the fermentation tank, which is not economical for production; in addition, according to the growth characteristics of microorganisms, too little inoculation will cause the mycelium to grow poorly or form mycelium balls, which will affect product synthesis. Therefore, the method of step-by-step expansion of culture is adopted, which is a summary of actual experience.
The determination of the level depends on the growth characteristics of the strain, the spore germination and bacterial reproduction rate, and the volume of the fermentation tank used. For fast-growing bacteria, the amount of seeds used is relatively small, and the number of stages is also relatively small. In the production of glutamate, eggplant bottle slant or shake bottle seeds are inoculated into seed tanks and cultured at 32°C for 7-10 hours. When the bacterial concentration reaches 10⁸-10⁹/mL, they can be inoculated into fermentation tanks as seeds. This is called primary seed tank expansion culture. The fermentation of primary seeds in fermentation tanks is called secondary fermentation. For slower growing strains, such as penicillin-producing strains, their spore suspension is inoculated into a first-level seed tank and cultured at 25°C for 40 hours. At this time, the spores germinate and mycelium grows. The suspension is then moved to a second-level seed tank containing fresh culture medium and cultured at 27°C for 15 to 24 hours. The mycelium rapidly reproduces and obtains robust bacteria. This mycelium can be moved to a fermentation tank as a seed. This is called secondary seed tank expansion culture and tertiary fermentation. Generally, 50m³ fermentation tanks use three-stage fermentation. For slower growing strains, such as Streptomyces griseus, three-stage seed tanks are generally used for expansion culture. When conducting experiments in small fermentation tanks (5~30L), direct spores or mycelium are also used for fermentation, which is called primary fermentation.
The fewer the number of seed tanks, the easier it is to simplify the process and control, and reduce the probability of contamination due to multiple transplants. However, the characteristics of each production strain and the equipment configuration of the production workshop must also be considered to improve the production efficiency of the fermentation tank. Although the number of seed tanks is determined by the product variety and production scale, it is also related to the selected process conditions. If the culture conditions of the seed tank are changed to accelerate the germination of spores and the reproduction of bacteria, the number of seed tanks can be reduced accordingly.