Feeding technology plays a very important role in the modern large-scale fermentation industry. Feeding can control the cell density and avoid the inhibitory effect of excessive substrate concentration on cell growth or cell expression.

At present, the feeding method has developed from simple one-stage feeding to multi-stage repeated feeding, from simply adding one nutrient to adding several nutrients, and the types of fed fermentation are also more diverse.

Ⅰ. Feeding fermentation

Fed-batch Fermentation refers to the intermittent or continuous addition of fresh medium containing limited nutrients during batch fermentation.

There was no fixed quantification concept in the early feeding methods, but a certain amount of nutrients were added at a certain time point based entirely on experience. This feeding method is very simple, and the ingredients and quantity of the feeding are relatively simple, but sometimes it is impossible to effectively control the fermentation.

Now there are more feeding methods, and the types of feeding can be roughly divided into the following types:

According to feeding method: continuous feeding, discontinuous feeding and multi-cycle feeding

According to the components of supplementary materials: complete supplementation (supplemented with complete medium) and semi-batch feeding (supplementation with one or several nutrients)

According to the number of reactors: single-stage and multi-stage

According to the volume change of the fermentation broth in the reactor: variable volume feeding and constant volume feeding

According to feeding control mode: feedback control feeding and non-feedback control feeding

Nutrients used for feeding generally fall into the following categories:

Supplement the energy and carbon sources needed by the bacteria, such as glucose and liquefied starch;

Supplement the nitrogen sources needed by the bacteria, such as organic nitrogen sources such as peptone, bean cake powder, corn steep liquor, yeast powder and urea; some fermentations also adopt ammonia gas or add ammonia water;

Add some trace elements and inorganic salts needed for growth and metabolism of bacteria, such as phosphate, sulfate, etc.;

For microorganisms that produce inducible enzymes, the substrates of the enzymes are often added to the feed to increase the production of the enzymes;

For the fermentation of some antibiotics, it is often necessary to supplement the precursors formed by the antibiotics.

Ⅱ. Feed control

Excessive or insufficient feeding will affect the growth of bacteria and the formation of products, and even lead to fermentation failure, so the control of feeding is a key point.

01 Feeding without feedback control

The flow rate of the added nutrients in feeding without feedback control is preset, and there are three modes of constant feeding, variable speed feeding and exponential feeding.

1. Constant speed feedingConstant-rate feeding feeds a limiting nutrient at a pre-set constant rate. Relative to the bacteria, the nutrient concentration decreased gradually, and the specific growth rate of the bacteria also decreased, while the total amount of the bacteria increased linearly. The constant feeding rate met the nutrient needs of the bacteria to a certain extent and avoided nutrient inhibition.

2. Variable speed feedingVariable-speed feeding means that the feeding rate is continuously increased in a gradient, stage or linear manner during the culture process. It can add more nutrients to promote the growth of cells when the concentration of bacteria is high, and achieve a continuous increase in the specific growth rate of cells, which is conducive to the formation of products, which is better than constant-rate supplementation.

3. Index feedingThe feeding rate of exponential feeding increases exponentially, which can control the nutrient concentration in the reactor at a low level, keep the specific growth rate of the bacteria at a constant value, and the density of the bacteria increases exponentially. Exponential feeding can better match the growth process of the bacteria, and does not require particularly complicated instruments, so it has received more attention.

02 Feedback Control Feeding

Feedback control feeding is the real-time or online detection and control of parameters such as nutrient concentration, product concentration and cell concentration in the reactor during the fermentation process.

The direct measurement parameters are temperature, pH, dissolved oxygen concentration, optical density, nutrient concentration, pressure and tail gas composition, etc., which can be directly measured by instruments and equipment.

Indirect measurement parameters include specific growth rate, bacterial cell concentration, oxygen uptake rate, oxygen transfer rate and carbon dioxide growth rate, etc., which can be evaluated or calculated by one or more direct measurement parameters.

1. Simple feedback control (single cycle method) feedingThis method controls parameters such as pH or dissolved oxygen concentration coupled with nutrient utilization to keep them constant. For example, if the pH is set at a constant value in advance, acidic substances or ammonium are produced during fermentation, which changes the pH value, thereby activating the control switch and feeding, and the pH returns to a constant value.

2. Nutrient intake or demand to control feedingNutrients are controlled within the set value by feeding, and the object of control is often the glucose concentration. For example, using intermittent feeding to ferment natamycin in batches, and intermittent sugar supplementation during fermentation to maintain the glucose concentration at 2%, the production of natamycin can be increased by 35%.

3. Control feeding according to specific growth rateThe greater the specific growth rate μ of the bacteria, the faster the microorganisms grow and consume more nutrients. For example, the oxygen uptake rate data is used to calculate the specific growth rate μ, and the glucose supplementation rate is controlled according to μ to obtain high-yield penicillin.

4. Analysis of tail gas composition to control feedingUtilization of nutrients is usually accompanied by the release of gases such as CO2. Measuring outlet gases, similar to analyzing nutrients, can also be used to control feed rates. This control method is relatively simple and can be used for the control of aerogenic fermentation, but it is easy to lag.

5. Control feeding according to cell morphologyIn the cultivation of some microorganisms, the morphological changes of cells are closely related to the culture conditions (such as dissolved oxygen concentration, shear rate, and medium composition), which requires an image sensor to detect the morphological changes of cells in the fermentation broth.

6. Feeding of fuzzy controlIt is difficult to describe the exact mathematical relationship between the measurable parameters in fermentation and the growth and metabolism of organisms, but it seems more appropriate to use membership functions to describe their fuzzy relationship. When conventional detection and control methods are difficult to accurately reflect the operating state of the system, fuzzy control theory is more unique.

7. Neural network control feedingArtificial neural network control is to simulate the operating mechanism of the human brain, using processing units instead of neurons, and relying on the interrelated information stored in the processing units to achieve control. This model is used to predict whether the fermentation state is normal, to predict the metabolic state, product, nutrient concentration, and the generation of various inhibition states.

Ⅲ. The advantages of feeding

 
1. It is conducive to the high-density cultivation of bacteria

The biomass of high-density fermentation can reach 60-150g/L, and it needs to input 2-5 times the nutrients of the biomass. If all the supplements are added to the medium at one time, excessively high concentration of nutrients will inevitably cause the disorder of bacterial metabolism, which is manifested as prolonged lag phase, lower specific growth rate, and lower yield. Therefore, it is necessary to adopt a suitable feeding method. .

2. It is conducive to the high-density cultivation of bacteria

Some fermentations require the use of toxic substances such as methanol, acetic acid, and phenol as media components, which can inhibit microbial growth even at low concentrations. And by feeding, the inhibitory effect can be reduced.

3. Release the repression caused by high-concentration nutrients

Glucose catabolites repress the synthesis of enzymes including cellulase, protease, amylase, invertase, and amino acid synthase. The growth rate of the bacteria is controlled by feeding to make the synthesis of the enzyme obviously derepressed; the adverse effects of ethanol, formic acid, lactic acid and other by-products produced by catabolism on the growth of the bacteria can also be reduced by feeding.

4. Maintain favorable fermentation conditions

The pH value often changes during the fermentation process. The pH value can be adjusted quickly by directly adding acid or alkali, while the pH value can be adjusted slowly and fundamentally by adding carbon or nitrogen sources. For aerobic fermentation, too much sugar at one time will cause the cells to grow too fast and consume oxygen rapidly, while conventional ventilation and stirring cannot meet the demand for oxygen supply, feeding can alleviate this contradiction.

In addition, feeding can also adjust the physical parameters such as the viscosity of the fermentation broth and the oxygen transfer coefficient, improve the fermentation environment, and be beneficial to cell growth and product synthesis.