Fed-batch technology is very important in modern large-scale fermentation industry. You can control microbial cell density with fed-batch operation. You can also avoid the inhibitory effect of too high substrate concentration on microbial growth or target product expression.
Now, fed-batch modes have changed. They were simple one-stage feeding before. Now they are multi-stage repeated feeding. They also changed from adding one nutrient to adding multiple nutrients. So there are more types of fed-batch fermentation.
I. Fed-Batch Fermentation
Fed-batch Fermentation means adding fresh culture medium with limiting nutrients off and on or continuously during batch fermentation.
In the early stage, fed-batch operation had no fixed quantitative rules. It only relied on experience. People added a certain amount of nutrients at certain time points. This method was very simple. The components and dosage of the added materials were relatively single. But it sometimes could not control the fermentation process well.
Now, fed-batch modes are more varied. They can be roughly divided as follows:
By feeding mode: Continuous feeding, discontinuous feeding and multi-cycle feeding
By composition of added materials: Complete feeding (adding full culture medium) and semi-batch feeding (adding one or several nutrients)
By number of bioreactors: Single-stage and multi-stage
By volume change of fermentation broth in bioreactors: Variable-volume feeding and constant-volume feeding
By feeding control mode: Feedback-controlled feeding and non-feedback-controlled feeding
The nutrients used for fed-batch operation are roughly divided into the following types:
Energy and carbon sources for microbial growth, such as glucose and liquefied starch;
Nitrogen sources for microbial growth, including organic nitrogen sources like peptone, soybean cake powder, corn steep liquor, yeast extract and urea; some fermentation processes also use ammonia gas or add ammonia water;
Trace elements and inorganic salts needed for microbial growth and metabolism, such as phosphate and sulfate;
For microorganisms that produce inducible enzymes, people add the enzyme’s substrate to the feed properly. This can increase enzyme yield;
For some antibiotic fermentations, people often need to add precursors for antibiotic synthesis.
II. Control of Fed-Batch Operation
Too much or too little feeding will affect microbial growth and product formation. It may even cause fermentation failure. So controlling fed-batch operation is a key point.
01 Non-Feedback-Controlled Feeding
The nutrient flow rate in non-feedback-controlled feeding is set in advance. There are three modes: constant-rate feeding, variable-rate feeding and exponential feeding.
- Constant-rate feeding
Limiting nutrients are added at a fixed rate set in advance. For microorganisms, the nutrient concentration goes down slowly. The specific growth rate of microorganisms also goes down. But the total microbial biomass increases in a straight line. Constant-rate feeding meets the nutrient needs of microorganisms to some degree. It also avoids nutrient inhibition.
- Variable-rate feeding
The feeding rate goes up continuously during cultivation. It can go up in ways like gradient, stage or linearity. When the cell concentration is high, it can add more nutrients. This promotes cell growth. It makes the specific cell growth rate go up continuously. This helps product formation. It is better than constant-rate feeding.
- Exponential feeding
The feeding rate goes up exponentially. It can keep the nutrient concentration in the bioreactor at a low level. It also keeps the specific growth rate of microorganisms stable. And it makes microbial cell density go up exponentially. Exponential feeding matches the microbial growth process well. It does not need very complex instruments. So it gets a lot of attention.
The feeding rate increases exponentially, which can control the nutrient concentration in the bioreactor at a low level, keep the specific growth rate of microorganisms constant, and realize the exponential increase of microbial cell density. Exponential feeding matches the microbial growth process well and does not require particularly complex instruments, thus attracting extensive attention.
02 Feedback-Controlled Feeding
Feedback-controlled feeding involves the real-time or on-line detection and control of parameters such as nutrient concentration, product concentration and cell concentration in the bioreactor during fermentation. Directly measured parameters include temperature, pH, dissolved oxygen concentration, optical density, nutrient concentration, pressure and off-gas composition, all of which can be directly measured by instruments and equipment.
Indirectly measured parameters include specific growth rate, microbial cell concentration, oxygen uptake rate, oxygen transfer rate and carbon dioxide production rate, which can be evaluated or calculated by one or more directly measured parameters.
- Simple feedback-controlled feeding (single cycle method)
This method controls parameters coupled with nutrient utilization, such as pH or dissolved oxygen concentration, to keep them constant. For example, a constant pH value is preset; during fermentation, acidic substances or ammonia produced by microbial metabolism cause changes in pH value, thereby activating the control switch to start feeding until the pH returns to the constant value.
- Feeding control based on nutrient uptake or demand
Nutrients are controlled within a preset range through feeding, with glucose concentration as the common control target. For instance, in the batch fermentation of natamycin with intermittent feeding, intermittent glucose supplementation to maintain the glucose concentration at 2% can increase the natamycin yield by 35%.
- Feeding control based on specific growth rate
A higher specific growth rate μ of microorganisms indicates faster microbial growth and more nutrient consumption. For example, the specific growth rate μ is calculated using oxygen uptake rate data, and the glucose feeding rate is controlled according to μ, resulting in high penicillin yield.
- Feeding control based on off-gas composition analysis
Nutrient utilization is usually accompanied by the release of gases such as CO₂. Determining the off-gas composition, similar to nutrient analysis, can also be used to control the feeding rate. This control method is relatively simple and applicable to the control of gas-producing fermentation, but it is prone to time lag.
- Feeding control based on cell morphology
In the cultivation of some microorganisms, changes in cell morphology are closely related to culture conditions (such as dissolved oxygen concentration, shear rate and medium composition), which requires image sensors to detect the morphological changes of cells in the fermentation broth.
- Fuzzy control for feeding
It is difficult to describe the relationship between measurable parameters and biological growth and metabolism in fermentation with an exact mathematical model, while describing their fuzzy relationship with membership functions seems more appropriate. Fuzzy control theory has its unique advantages when conventional detection and control methods cannot accurately reflect the operating state of the system.
- Neural network control for feeding
Artificial neural network control simulates the operation mechanism of the human brain, uses processing units to replace neurons, and realizes control by relying on the interrelated information stored between processing units. This model is used to predict whether the fermentation state is normal, as well as metabolic state, product and nutrient concentrations, and the occurrence of various inhibitory states.
III. Advantages of Fed-Batch Operation
- Facilitating high-density microbial cultivation
The biomass of high-density fermentation can reach 60-150g/L, requiring 2-5 times the amount of nutrients relative to the biomass. If all nutrients are added to the medium at one time, the excessively high nutrient concentration is bound to cause disorder of microbial metabolism, manifested as prolonged lag phase, reduced specific growth rate and decreased yield. Therefore, an appropriate fed-batch mode is required.
- Reducing the inhibitory effect of toxic substrates
Some fermentation processes use toxic substances such as methanol, acetic acid and phenol as medium components, which can inhibit microbial growth even at low concentrations. Fed-batch operation can reduce such inhibitory effects.
- Relieving the catabolite repression caused by high-concentration nutrientsGlucose catabolites can repress the synthesis of enzymes including cellulase, protease, amylase, invertase and amino acid synthetase. Controlling the microbial growth rate through fed-batch operation can significantly derepress enzyme synthesis; fed-batch operation can also reduce the adverse effects of by-products such as ethanol, formic acid and lactic acid produced by catabolism on microbial growth.
- Maintaining favorable fermentation conditions
pH changes often occur during fermentation. Direct addition of acid or alkali can adjust the pH rapidly, while supplementation of carbon or nitrogen sources can adjust the pH slowly and fundamentally. For aerobic fermentation, excessive one-time sugar addition will cause rapid cell growth and oxygen consumption, which cannot be met by conventional aeration and agitation. Fed-batch operation can alleviate this contradiction. In addition, fed-batch operation can adjust the physical parameters of the fermentation broth such as viscosity and oxygen transfer coefficient, improve the fermentation environment, and facilitate cell growth and product synthesis.
IV. Conclusion
With the in-depth understanding of microbial physiology and biochemistry, the improvement of sensing technology, and the continuous development of computers and modern control theory, fed-batch fermentation technology will surely undergo greater improvements and be applied more extensively in the industry.
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