In E. coli fermentation, the feeding rate and the specific growth rate directly affect acetic acid production and buildup because they change the amount of residual sugar in the broth. So, by controlling the feeding amount and the specific growth rate, acetic acid can be controlled in E. coli fermentation.
You must ensure enough dissolved oxygen in E. coli fermentation, and you must control the pH strictly. Acid and alkali should be added slowly and evenly. Temperature strongly affects protein expression in E. coli fermentation. At low temperature, fermentation mainly produces active proteins. At high temperature, fermentation mainly produces inclusion bodies.
Induction should be done in the late exponential growth phase, and the specific growth rate should be kept below 0.2. This timing has clear benefits. First, bacterial growth and protein synthesis are separated, so they do not disturb each other, and protein production is increased. Second, the biomass is already high and remains almost stable. This improves process efficiency and reduces energy use and cost.
The carbon–nitrogen ratio during feeding is very important in E. coli fermentation. If there is too much nitrogen, bacteria grow too fast and the pH rises, which is not good for metabolite production. If nitrogen is too low, bacterial growth is weak and the yield is low. If carbon is too high, the pH drops and growth is inhibited. If carbon is too low, bacteria age quickly and may break down. An unbalanced carbon–nitrogen ratio also causes poor nutrient uptake, and this affects both growth and product formation in E. coli fermentation.
In practice, if bacterial lysis always occurs at a fixed time in an otherwise stable fermentation, and phage infection or strain problems are excluded, then the cause is often an improper carbon–nitrogen ratio. In this case, adjusting the ratio can improve the E. coli fermentation.process.
一、By-products, Temperature, and Culture Methods
Control of the Metabolic By-product Acetic Acid
Acetic acid is a major by-product in E. coli fermentation. People do not fully agree on the acetic acid level that strongly limits bacterial growth in E. coli fermentation. However, many studies show that under good aeration, 5–10 g/L acetic acid clearly lengthens the lag phase, lowers the maximum growth rate, reduces cell density, and decreases protein yield. When acetic acid reaches 10–20 g/L, bacterial growth stops. When it is above 12 g/L, foreign protein expression stops.
Ways to reduce acetic acid
- Slow growth to reduce acetic acid
When bacteria grow faster, they produce more acetic acid. If the growth rate is too high, acetic acid builds up. Growth can be slowed by lowering temperature, adjusting pH, or controlling feeding. - Dialysis culture
During fermentation, dialysis can be used to remove harmful substances, including acetic acid. This supports high cell density and improves protein production in recombinant strains. - Control glucose concentration
Glucose is a main carbon source. So, its level should be kept low to reduce acetic acid formation.
When acetic acid reaches 10–20 g/L, bacterial growth stops. When the acetic acid level is higher than 12 g/L, foreign protein expression almost stops completely.
Common Control Methods
1、Constant pH method
E. coli breaks down glucose and produces acetic acid in E. coli fermentation, which lowers the pH. Because of this, pH can be used as a signal to add glucose. However, pH changes are not caused only by glucose metabolism, so this method can sometimes give wrong feeding signals.
2、Constant dissolved oxygen method
Bacteria consume oxygen during metabolism in E. coli fermentation, so the dissolved oxygen level goes down. When glucose becomes very low, bacterial metabolism slows down, and the dissolved oxygen level rises.
They use less oxygen, so the dissolved oxygen level goes up. So, you can add glucose based on the dissolved oxygen curve. Keeping dissolved oxygen constant helps keep glucose at the right level in E. coli fermentation.
二、Temperature
The best temperature for E. coli to grow is 37 °C. At this temperature, the bacteria grow the fastest. Higher temperatures make the bacteria’s metabolism faster in E. coli fermentation. But they also make more harmful by-products. Fast growth can also make the plasmid less stable. On the other hand, lower temperatures make the bacteria take in fewer nutrients. They grow more slowly. This also reduces the amount of toxic by-products and metabolic heat. Sometimes, lower temperatures help the target protein fold correctly and be produced well.
When fermenting recombinant E. coli, the best temperature is different for different phases. To get a lot of target protein, you first need to have a lot of bacteria. So, in the early phase, you should focus on growing the bacteria. In the induction phase, you should focus on producing the target product.
三、Culture Methods
Most E. coli fermentations use fed-batch culture. This is a way to improve modern fermentation processes. It helps make the chemical environment good for the microbes. It keeps the microbes in the best condition for growth. This method has two advantages.
First, it avoids the problem of substrate inhibition. This problem happens when some nutrients are too concentrated at the start.
Second, it stops limiting nutrients from running out. If these nutrients run out, the bacteria cannot grow well and the product cannot be made. Fed-batch culture is widely used in fermenting various primary and secondary biological products, as well as proteins，in E. coli fermentation.
About Ferbio
Ferbio has a lot of experience in making various bioreactors and pressure vessels. We have a team of experts. They know about bioreaction, fermentation technology, mechanical manufacturing, and automation control. Our research and technology are among the best in China and are at an advanced level in the world. We provide customers with reliable and satisfying product experiences.