First of all, the feeding rate and specific growth rate directly affect the production rate and accumulation of acetic acid (mainly because the feeding rate and specific growth rate affect the amount of residual sugar in the fermentation broth, thereby affecting). Therefore, proper control of the feed amount and specific growth rate has a good effect on controlling acetic acid.

Secondly, it is necessary to ensure sufficient dissolved oxygen, strictly control the pH value, and supplement acid and alkali at a moderate rate, not too fast; temperature also has an important impact on protein expression. Most of the proteins produced by low-temperature fermentation are active, while most of the proteins produced by high-temperature fermentation exist in the form of inclusion bodies.

Third, it is very important to choose a reasonable induction time. Generally, the induction time is selected in the late stage of exponential growth, and the specific growth rate during induction is preferably controlled within 0.2. Induced at this time:

1 Separating the rapid growth phase from the protein synthesis phase, the two phases do not affect each other, which is conducive to high protein expression.

2 A large number of bacterial cells were obtained, and the biomass of the bacterial cells was basically close to stable, which was reasonable from the perspectives of kinetics, energy consumption and material cost.

Fourth, the carbon-nitrogen ratio during the feeding process is also important. If the nitrogen source is too high, the bacteria will grow vigorously, and the pH value will be too high, which is not conducive to the accumulation of metabolites. If the nitrogen source is insufficient, bacterial growth will be less, affecting yield. If there are too many carbon sources, it is easy to cause a low pH and inhibit the growth of bacteria. If the carbon source is insufficient, it is easy to cause aging and autolysis of the bacteria. In addition, an improper carbon-nitrogen ratio will also cause an imbalance in the proportion of nutrients absorbed by bacteria, directly affecting bacterial growth and product synthesis.

According to experience, in general, for a stable fermentation process, if lysis always occurs during a fixed fermentation period, the possibility of phages and bacteria can be ruled out, and it may be caused by an unreasonable carbon-nitrogen ratio. The carbon-nitrogen ratio can be adjusted appropriately.

By-products, temperature and culture methods

◆Control of metabolic by-product-acetic acid

Acetic acid is a metabolic by-product during the fermentation process of E. coli. There have been different views on what concentration of acetic acid can produce an inhibitory effect. It is generally believed that under good gas conditions, an acetic acid concentration of 5-10g/L can have a significant inhibitory effect on the lag phase, maximum specific growth rate, bacterial concentration and final protein yield. When the acetic acid concentration is greater than 10 or 20g/L, cell growth stops. When the acetic acid concentration in the culture medium is greater than 12g/L, the expression of exogenous proteins is completely inhibited.

Measures to prevent the production of acetic acid:

1 Reduce acetic acid production by controlling growth rate:

The higher the specific growth rate, the more acetic acid is produced. When the specific growth rate exceeds a certain value, acetic acid will be produced. The growth rate can be reduced by lowering the temperature, adjusting the pH value, and controlling feeding.

2 Dialysis culture:

During the cultivation of E. coli, dialysis technology can be used to remove harmful substances in the fermentation broth and reduce the acetic acid content, thereby achieving high-density fermentation and product expression of the recombinant bacteria.

3 Controlling glucose concentration:

Glucose is one of the important carbon sources in the fermentation process of E. coli. The purpose of using glucose as a carbon source is to control it at low levels to reduce the production of acetic acid.

◆Commonly used control methods mainly include:

Constant pH method:

E. coli will metabolize grapes and produce acetic acid, which will lower the pH value. Therefore, the pH value can be used as an indicator to control the amount of sugar. The disadvantage of this method is that changes in pH are not entirely the result of sugar metabolism and can easily cause errors in the feeding system.

Constant dissolved oxygen method:

The metabolism of bacteria will consume oxygen, reducing the amount of dissolved oxygen. When the glucose concentration reaches a certain level, bacterial metabolism will decrease, oxygen consumption will decrease, and dissolved oxygen will increase. Therefore, by adding glucose according to the dissolved oxygen curve and keeping the dissolved oxygen constant, glucose can be controlled at a certain level.

◆Temperature

The optimal temperature for E. coli fermentation is 37 degrees Celsius. When the temperature is most suitable for bacterial growth, the specific growth rate will increase. As temperature increases, bacterial metabolism accelerates and the production of metabolic by-products increases. These by-products can inhibit the growth of bacteria to a certain extent. Plasmid stability can also be affected by rapid bacterial growth. When culture temperatures are lowered, nutrient uptake and bacterial growth rates decrease. It also reduces the production of toxic metabolic by-products and metabolic heat. Sometimes lowering the temperature is more conducive to the correct folding and expression of the target protein.

In the fermentation process of recombinant E. coli, the optimal temperatures at different fermentation stages are different. In order to obtain a large amount of target protein, we must first ensure the number of bacterial cells. Therefore, priority can be given to cultivating bacterial strains in the early stage, and priority in expressing the target product in the induction stage.

◆Training method

Most E. coli fermentations use fed-batch culture, which is a way to optimize modern fermentation processes and effectively optimize the chemical environment during microbial culture. Keep microorganisms in the best growth environment.

On the one hand, this method can avoid substrate inhibition when the initial concentration of certain nutrients is too high, and on the other hand, it can prevent the depletion of limiting nutrients from affecting cell growth and product formation. Fed-batch culture has been widely used in the fermentation of various primary and secondary biologics and proteins.