Selection of culture medium
Different microorganisms have different requirements for culture medium, so different microbial culture processes have different requirements for raw materials. According to the specific situation, starting from the characteristics of microbial nutrition demand and the requirements of production technology, select the appropriate nutrient medium, so that it can not only meet the needs of microbial growth, but also obtain high-yield products, and at the same time conform to the principle of increasing production and saving, adapting to local conditions.
1, and the medium is selected according to the characteristics of microorganisms.
Microorganisms used for large-scale culture mainly include bacteria, yeasts, molds and actinomycetes. They have different requirements for nutrients and have their own characteristics. In practical application, the composition of culture medium should be considered according to the different characteristics of microorganisms, and the formula of typical culture medium should be adjusted as necessary.
2. Select the culture medium according to the fermentation mode.
Liquid and solid media have their own uses and advantages and disadvantages. In liquid culture medium, nutrients are dissolved in water in solute state, so that microorganisms can fully contact and utilize nutrients, which is more conducive to the growth of microorganisms and the better accumulation of metabolites. In industry, submerged fermentation with liquid culture medium has the advantages of high fermentation efficiency, convenient operation, convenient mechanization and automation, reduced labor intensity, small floor space and high yield. Therefore, in the fermentation industry, most seeds are cultured and fermented in liquid medium, and according to the oxygen demand of microorganisms, they are cultured in static or aeration mode respectively. Solid culture medium is often used for the preservation, separation, colony characteristics identification and living cell counting of microbial strains. In addition, some solid raw materials, such as millet, rice, bran, potatoes and so on. It is commonly used in industry to directly make inclined planes or eggplant bottles to cultivate molds and actinomycetes.
3. Different requirements from production practice and scientific experiments.
In the production process, due to the different purposes and requirements of various stages such as strain preservation, seed expansion culture and fermentation production, the proportion of components in the selected culture medium should also be different. Generally speaking, seed culture medium is mainly used for the growth and proliferation of microbial cells. In order to obtain a large number of robust seed cells in a short time, the seed culture medium should be nutritious and complete, the ratio of nitrogen source to vitamin should be high, and the raw materials used should be easily absorbed and utilized by microbial cells. Glucose, ammonium sulfate, urea, corn syrup, yeast extract, wort and rice koji juice are common raw materials for preparing culture medium. In addition to maintaining the normal growth of microbial cells, fermentation medium mainly needs to synthesize predetermined fermentation products, so the content of carbon source substances in fermentation medium is often higher than that in seed medium. Of course, if the product is a nitrogen-containing substance, the supply of nitrogen source should be increased accordingly. In addition, the fermentation medium should be considered to be convenient for fermentation operation and not affect the extraction and separation of products and product quality.
4. Select raw materials for production from the perspective of economic benefits.
From a scientific point of view, the economy of culture medium is usually ignored. For the production process, the raw materials for preparing fermentation medium are mostly grain, oil, protein and so on. However, industrial fermentation consumes a lot of raw materials, which are easy to be used by microorganisms and meet the requirements of production technology. In addition, economic benefits should also be considered when selecting culture medium raw materials in industrial fermentation, and it must be based on the premise of low price, abundant sources, convenient transportation, local materials and non-toxicity.
Principles of medium preparation
The preparation of culture medium must provide the basic components of synthetic microbial cells and fermentation products; It is beneficial to reduce the unit consumption of culture medium raw materials, and the number of products synthesized by unit nutrients is large or the output is large; It is beneficial to improve the concentration of culture medium products, thus improving the production capacity of unit volume fermentor; It is beneficial to improve the synthesis speed of products and shorten the fermentation period; Minimize the formation of by-products; Reducing the influence of fermentation on aeration and stirring is beneficial to improve oxygen utilization and reduce energy consumption; Conducive to the separation and purification of products; And minimize the substances that produce "three wastes".
Of course, it is impossible to design any kind of culture medium to meet all the above requirements, and it is necessary to grasp the main links according to the specific situation. It can not only meet the nutritional requirements of microorganisms, but also obtain high-quality and high-yield products, and also conform to the principle of increasing production and saving, adapting to local conditions. The main function of fermentation medium is to obtain the expected product, and the medium must be designed according to the characteristics of the product. Therefore, nutrition should be rich and complete, bacteria should grow rapidly and healthily, and the pH value of the whole metabolic process should be appropriate and stable. The metabolism of sugar and nitrogen can completely meet the requirements of high-unit tanks and batches, and can give full play to the ability of the strain to produce synthetic metabolites. In addition, it is necessary to reduce costs.
1, different culture media are prepared according to the nutritional requirements of different microorganisms.
Different microorganisms need different medium components. To determine the appropriate culture medium, it is necessary to know the source, physiological and biochemical characteristics and general nutritional requirements of the producing strains, and determine the culture medium according to the culture conditions of different producing strains, metabolic pathways of biosynthesis and chemical properties of metabolites.
2. Appropriate proportion of nutrients
Microorganisms need a proper proportion of nutrients, and the ratio of carbon to nitrogen (C/N) in culture medium is particularly important in fermentation industry. Different microbial strains and different fermentation products need different C/N ratios. At different growth stages, the optimum requirements for carbon-nitrogen ratio are different. The ratio of carbon to nitrogen in culture medium will not only affect the growth of microbial cells, but also affect the metabolic pathway of fermentation. Because carbon is both a carbon frame and an energy source, it is used more than nitrogen. According to the elemental analysis, the carbon-nitrogen ratio of yeast cells is about 100:20, and that of mold cells is about 100: 10. Generally speaking, the ratio of carbon to nitrogen in fermentation medium is about 100: (0.2 ~ 2.0), but in amino acid fermentation, the ratio of carbon to nitrogen is relatively high because the product contains nitrogen. For example, the ratio of carbon to nitrogen in glutamic acid fermentation is100: (15 ~ 21). If the ratio of carbon to nitrogen is 100: (0.2 ~ 2.0), only bacteria will grow and almost no glutamic acid will be produced.
The ratio of carbon to nitrogen varies with the types of carbohydrates and nitrogen sources and the conditions of aeration and stirring, so it is difficult to determine a unified ratio. Under normal circumstances, the low ratio of carbon to nitrogen will lead to the vigorous growth of bacteria, which will easily lead to premature aging and autolysis of bacteria and affect the accumulation of products; The ratio of carbon to nitrogen is too large, and the number of bacteria is small, which is not conducive to the accumulation of products; Carbon and nitrogen are suitable, but high concentration of carbon and nitrogen can still lead to a large number of cell proliferation, increase the viscosity of fermentation broth, affect the dissolved oxygen concentration, easily cause abnormal cell metabolism and affect product synthesis; Carbon and nitrogen sources are suitable, but the low concentration of carbon and nitrogen sources will affect the reproduction of bacteria and the accumulation of products.
3. Osmotic pressure
When preparing culture medium, we should pay attention to the proper concentration of nutrients. The concentration of nutrients is too low, which not only can not meet the demand of microbial growth for nutrients, but also is not conducive to improving the yield of fermented products and improving the utilization rate of equipment. However, when the concentration of nutrients in the culture medium is too high, the growth of microorganisms will be inhibited due to the high osmotic pressure of the culture medium solution. In addition, the concentration ratio of various ions in the culture medium will also affect the osmotic pressure of the culture medium and the metabolic activities of microorganisms. Therefore, the proportion of various ions in the culture medium should be balanced. In the process of fermentation production, under the condition of not affecting the physiological characteristics and metabolic conversion rate of microorganisms, it is usually inclined to ferment at a higher concentration to improve the product yield, and the production strain with high osmotic pressure should be selected as much as possible. Of course, if the medium concentration is too high, the viscosity of the medium will increase and the dissolved oxygen will decrease.
4.ph value
The normal growth of various microorganisms needs a suitable pH value. Generally, mold and yeast are more suitable for weak acid environment, while actinomycetes and bacteria are suitable for neutral or slightly alkaline environment. Therefore, when preparing the culture medium, if the pH value is not suitable, it must be adjusted. When microorganisms change the pH value of the culture medium in the process of culture, which is not conducive to their own growth, the composition of the culture medium should be considered according to the utilization rate of various nutrients by microbial cells, and buffer solution should be added to adjust the pH value of the culture medium.
5. Redox potential
For most microorganisms, the redox potential of the culture medium generally has little effect on their growth, that is, the redox potential suitable for their growth is wide. However, for anaerobic bacteria, due to the toxic effect of oxygen, reducing agents are often added to the culture medium to reduce the redox potential.
When preparing culture medium, we should not only pay attention to the above principles, but also consider the order of adding nutrients. In order to avoid the loss of nutrients caused by precipitation, the order of adding nutrients is generally to add buffer compounds first, then add main substances after dissolution, and then add auxins such as vitamins and amino acids.