1. Natural selection of microorganisms
This refers to the process of selecting superior strains during production without artificial treatment, by taking advantage of the spontaneous mutations of the strains. Spontaneous mutations of strains often have two possibilities: strain degeneration and decreased production performance; or more vigorous metabolism and increased production performance. Experienced and observant staff can select superior strains based on the changes in strain characteristics that occur due to spontaneous mutations. For example, in the fermentation process of glutamic acid, strains resistant to bacteriophages were isolated from the contaminated fermentation broth. Another example is in the production of antibiotics, where high-yielding strains can often be obtained by sampling and isolating from a batch of high-yielding fermentation broth. However, the frequency of spontaneous mutations is relatively low, and the possibility of obtaining superior strains is small. It requires a considerable amount of time to achieve results.
2. Mutagenesis breeding of microorganisms
Mutagenesis breeding refers to the artificial treatment of microorganisms to induce mutations, and then screening out the mutant strains that meet the requirements for use in production and scientific experiments. Compared with other breeding methods, mutagenesis breeding has the advantages of simplicity, speed, and high efficiency. It remains an important and widely used method for microbial breeding. Mutagenesis breeding includes three parts: selection of the starting strain, mutagenesis treatment, and screening of mutant strains.
After mutagenesis treatment of the strains, various types of mutations will occur. How to select the desired mutant types? Generally, it involves two stages: primary screening and secondary screening. Let's take the screening of high-yielding mutant strains of penicillin-producing bacteria as an example. The mutagenized bacterial liquid is diluted to a certain concentration and spread on solid medium plates. After cultivation, individual colonies are picked onto slant medium for further cultivation. Then, each colony on the slant medium is inoculated into shake flasks for shaking cultivation, and the antibiotic titer is measured. This is the primary screening. Strains with titer exceeding 10% of the control in the primary screening are subjected to secondary screening. The process of secondary screening is basically the same as that of primary screening, except that individual colonies on the slant medium are usually inoculated into three shake flasks to obtain the average titer. Secondary screening can be conducted 1 to 3 times. The high-yielding and stable strains screened out still need to undergo small-scale or even medium-scale tests before being used in fermentation production.
1. Raw materials: carbon source, nitrogen source, growth factors, inorganic salts, and water.
2. Principles: clear objectives, balanced nutrition, and appropriate pH.
There should be no contamination by foreign bacteria during the fermentation process. Not only the culture medium but also the fermentation equipment and the air introduced need to be sterilized. Sterilization should not only kill foreign bacteria but also spores and spores.
Enlargement culture can shorten the adjustment period of microbial growth.
This is the central stage of fermentation. The key is to control the fermentation conditions, such as temperature, pH, dissolved oxygen, aeration rate, and rotation speed. The reason is that changes in environmental conditions not only affect the growth and reproduction of the strains but also the formation of their metabolic products. Factors affecting the fermentation processThe main factors affecting the fermentation process are as follows.
1. Temperature control
Temperature has multiple effects on microorganisms. First, temperature affects enzyme activity. Within the optimal temperature range, as the temperature increases, the growth and metabolism of the microorganisms accelerate, and the rate of the fermentation reaction increases. When the temperature exceeds the optimal range, as it rises, enzymes quickly lose activity, the microorganisms age, the fermentation cycle shortens, and the yield decreases. Temperature can also influence the biosynthetic pathways. For example, Streptomyces aureofaciens has a stronger ability to synthesize aureomycin at temperatures below 30°C, but when the temperature exceeds 35°C, it only synthesizes tetracycline and not aureomycin. In addition, temperature also affects the physical properties of the fermentation broth and the decomposition and absorption of nutrients by the microorganisms. Therefore, to ensure a normal fermentation process, it is necessary to maintain the optimal temperature. However, the optimal temperature for the growth of the microorganisms and the synthesis of products may not be the same. For example, the optimal growth temperature of Streptomyces griseus is 37 ℃, but the optimal temperature for the production of antibiotics is 28 ℃. Usually, the optimal temperature for different microorganisms at each stage of fermentation must be determined through experiments, and segmented control should be adopted.
2. Appropriate pH value
pH can affect the activity of enzymes and the charge status of cell membranes. If the charge status of the cell membrane changes, the permeability of the membrane will also change, which may affect the absorption of nutrients by microorganisms and the secretion of metabolic products. In addition, pH can also affect the decomposition of nutrients in the culture medium. Therefore, the pH of the fermentation broth should be controlled. However, the optimal pH for different microorganisms at different growth stages and product synthesis stages is often different and needs to be controlled separately. During the fermentation process, as the microorganisms utilize nutrients and accumulate metabolic products, the pH of the fermentation broth will inevitably change. For example, when urea is decomposed, the concentration of NH+4 in the fermentation broth will increase, and the pH will also increase. In industrial production, it is common to add a pH buffering system to the fermentation broth or control the pH by adding ammonia water, urea, ammonium carbonate or calcium carbonate during the process. Currently, pH electrodes for detecting the fermentation process have been developed in China, which can continuously measure and record pH changes, and the pH controller can adjust the addition of acid and alkali.
3. Dissolved oxygen
The supply of oxygen is a key factor for aerobic fermentation. From the perspective of the oxygen requirement for glucose oxidation, 1 mol of glucose requires 6 mol of oxygen for complete oxidation and decomposition; when glucose is used for the synthesis of metabolic products, about 1.9 mol of oxygen is needed for 1 mol of glucose. Therefore, aerobic microorganisms have a large demand for oxygen, but during the fermentation process, the microorganisms can only utilize the dissolved oxygen in the fermentation broth, and oxygen is difficult to dissolve in water. At 101.32 kPa and 25 ℃, the solubility of oxygen in water is 0.26 mmol/L. Under the same conditions, the solubility of oxygen in the fermentation broth is only 0.20 mmol/L, and it will decrease with the increase in temperature. Therefore, a large amount of oxygen must be continuously added to the fermentation broth and constant stirring should be carried out to increase the solubility of oxygen in the fermentation broth.
4. Foam
During the fermentation process, aeration and stirring, the metabolic process of microorganisms, and the decomposition of certain components in the culture medium may all produce foam. The production of a certain amount of foam during the fermentation process is a normal phenomenon, but excessive and persistent foam is detrimental to the fermentation. Because foam will occupy the volume of the fermentation tank, affect the normal aeration and stirring, and even lead to metabolic abnormalities, it must be eliminated. Common measures to eliminate foam include two types: one is to install foam-breaking baffles, which can break the foam through strong mechanical vibration; the other is to use defoaming agents.Concentration of nutrients The concentration of various nutrients in the fermentation broth, especially the carbon-nitrogen ratio, inorganic salts and vitamins, will directly affect the growth of microorganisms and the accumulation of metabolic products. For example, in glutamic acid fermentation, changes in the concentration of NH+4 will affect the metabolic pathway (see Glutamic acid fermentation). Therefore, during the fermentation process, the concentration of nutrients should also be controlled according to specific conditions. Taking the production of glutamic acid as an example, the regulation and control of oxygen, temperature, pH and phosphate during the fermentation process are as follows:
①Oxygen. Glutamic acid-producing bacteria are aerobic bacteria. Aeration and stirring not only affect the utilization rate of nitrogen and carbon sources by the microorganisms, but also affect the fermentation cycle and the synthesis of glutamic acid. Especially in the later stage of fermentation, increasing the aeration rate is beneficial to the synthesis of glutamic acid.
②Temperature. The optimal temperature for the growth of the strain is 30 to 32 ℃. When the bacteria reach the stable growth phase, a slight increase in temperature is beneficial for acid production. Therefore, in the later stage of fermentation, the temperature can be raised to 34 to 37 ℃.
③pH. The optimal pH for the fermentation of glutamic acid-producing bacteria is between 7.0 and 8.0. However, during the fermentation process, the pH of the culture medium will constantly change due to the utilization of nutrients and the accumulation of metabolic products. For instance, as the nitrogen source is utilized and ammonia is released, the pH will rise; when sugar is utilized to produce organic acids, the pH will drop.
④Phosphate. It is essential in the glutamic acid fermentation process, but the concentration should not be too high, otherwise it may lead to valine fermentation. After the fermentation is completed, ion exchange resin method and other techniques are commonly used for extraction.
Metabolic products: Distillation, extraction, ion exchange and other methods can be used for extraction.
The bacteria themselves: Filtration, precipitation and other methods can be used to separate them from the culture medium.
The production level of microbial fermentation mainly depends on the genetic characteristics of the strain itself and the culture conditions. The application scope of fermentation engineering includes:
1. Pharmaceutical industry,
2. Food industry,
3. Energy industry,
4. Chemical industry,
5. Agriculture: Plant gene modification; biological nitrogen fixation; engineering insecticidal bacteria biopesticides; microbial feed.
6. Environmental protection and other aspects. However, during the microbial fermentation process, it is crucial to prevent contamination. Once contaminated by other strains, the desired product cannot be produced.