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In this article we will discuss about Culture Media:- 1. Meaning of Culture Media 2. Components of Culture Media 3. Culture Procedures.
Meaning of Culture Media:
Media requirements depend on the type of microorganism being used in the fermentation process, but the basic requirements remain the same, which includes:
(a) Source of energy
(b) Water, carbon source
(c) Nitrogen source
(d) Vitamins
(e) Minerals.
Designing the media for small scale laboratory purpose is relatively easy, but media for industrial purpose are difficult to prepare.
An ideal culture medium has the following features:
(a) Allow high yield of the desired product and at fast rate
(b) Allow low yield of undesired products
(c) Be sterilized easily
(d) Yield consistent products i.e., minimum batch variation
(e) Be cheap and readily available
(f) Be compatible with the fermentation process
(g) Not pose environmental problems before, during, or after the fermentation process.
The culture medium will affect the design of the fermenter. For example, hydrocarbons in the media require high oxygen content, so an air-lift fermenter should be used. Natural media ingredients are cheap but they have high batch variation.
On the other hand pure ingredients (also called defined media or formulated media) have very little batch variation but are expensive. The media should support the metabolic process of the micro-organisms and allow bio-synthesis of the desired products.
Carbon & Energy source + Nitrogen source + Nutrients → Products(s) + Carbon Dioxide + Water + Heat + Biomass
Components of Culture Media:
Media are designed based on the above equation using minimum components required to produce maximum product yield.
Important components of the medium are discussed as follows:
1. Carbon Sources:
Product formation is directly dependent on the rate at which the carbon source is metabolized. Also the main product of fermentation determines the type of carbon source to be used. Carbon sources include carbohydrates, oils and fats, and hydrocarbons.
(a) Carbohydrates:
These are the most commonly used carbon sources in the fermentation process. Starch is easily available carbohydrate obtained from maize, cereals, and potatoes. It is widely used in alcohol fermentation. Grains like maize are used directly in the form of ground powder as carbohydrate.
Malt and beer made from barley grains contain high concentrations of different carbohydrates like starch, sucrose, cellulose and other sugars. Sucrose is obtained from sugar cane and molasses. Molasses is one of the cheapest sources of carbohydrate.
It contains high sugar concentration and other components like nitrogenous substances and vitamins and is used in alcohol, SCP (Single-cell Protein), amino acid, and organic acid fermentations. Extraction and purification of the products is expensive.
Sulfate waste liquor is the by-product of the paper industry; it contains carbohydrates and is used in yeast cultivation. Whey is the by-product of dairy industry. It is used in alcohol, SCP, gum, vitamins, and lactic acid fermentation.
(b) Oils and Fats:
Vegetable oils are used as a carbon source. Oils provide more energy per weight compared to sugars. They also have anti-foaming properties but are generally used as additives rather than as the sole carbon source. Examples are olive oil, cotton seed oil, soya bean oil, linseed oil, and lard (animal fat).
(c) Hydrocarbons:
C12-C18 alkanes can be used as carbon sources. They are cheap, and have more carbon and energy content per weight than sugars. They can be used in organic acids, amino acids, antibiotics, enzymes, and proteins fermentation.
2. Nitrogen Sources:
Ammonia, ammonium salts, and urea are the most commonly used nitrogen sources in the fermentation process. Ammonia also serves the purpose of pH control. Other substances used as nitrogen sources are corn-steep liquor, soya meal, peanut meal, cotton seed meal, amino acids, and proteins.
3. Minerals:
Calcium, chlorine, magnesium, phosphorous, potassium and sulfur are the essential minerals for all media. Other minerals like copper, cobalt, iron, manganese, molybdenum, and zinc are needed in trace amounts and are generally present as impurities in other components. The specific concentration on these elements depends on the micro-organism being used.
4. Growth Factors:
Vitamins, amino acids, and fatty acids are used as growth factors in the fermentation process to complement the cell components of the micro-organisms.
5. Chelating Agents:
Chelating agents prevent formation of insoluble metal precipitates. They form complexes with the metal ions present in the medium and can be utilized by the micro-organisms. Chelating agents are not required in large scale fermentation processes since some of the other ingredients like yeast extract will perform the function of forming complexes with the metal ions.
One example of a chelating agent is EDTA (ethylenedi-aminetetra-acetic acid). EDTA is a versatile, being able to form six bonds with a metal ion. It is frequently used in soaps and detergents because it forms a complex with calcium and magnesium ions.
These ions are in hard water and interfere with the cleaning action of soaps and detergents. Other chelating agents are citric acid and pyrophosphates
6. Buffers:
Buffers are used to maintain the pH of the medium as microbial growth is affected by the pH changes. Optimum pH for most microorganisms is 7.0. Commonly used buffers are calcium carbonate, ammonia, and sodium hydroxide.
7. Antifoaming Agents:
Microbial process produces a large amount of foam in the fermentation vessel. This is due to microbial proteins or other components of the media. Foaming causes removal of cells from the media and their autolysis, thus, releasing more microbial foam-producing proteins, hence, aggravating the problem. Foam will reduce the working volume in the fermentation vessel, decrease rate of heat transfer, and deposit cells on the top of the fermenter.
The air filter exits then become wet allowing growth of contaminating microorganisms. Antifoaming agents are also called surfactant, i.e., they reduce the surface tension in the foam and destabilize the foam producing proteins. Commonly used antifoaming agents are stearyl alcohol, cotton seed oil, linseed oil, olive oil, castor oil, soya bean oil, cod liver oil, silicones, and sulphonates.
8. Air:
Air is required for aeration and is supplied to the fermenter by means of pumps or compressors. It is sterilized by passing through filters before being introduced. The amount of air required and the extent of purity depends on the fermentation process being carried out.
9. Steam:
Steam is used to sterilize fermenters and other equipment and to control temperature. Continuous dry steam supply is required for the fermentation process, and care should be taken to prevent condensation.
Culture Procedures for Culture Media:
1. Sterilization:
The media and culture vessel are sterilized in order to prevent the growth of any unwanted microorganisms which contaminates our desired culture. Sterilization is carried out in many ways. The most widely method of sterilization is done by the help of an autoclave which is a type of steam sterilization.
An autoclave or pressure cooker maintains a temperature of 120°C for 15 to 20 minutes under 15 psi pressures. Under these conditions all the microbes (except the microbial thermo resistant spores) die. When microbes are cultivated in a fermentor in the industries then it is convenient to sterilize the fermentor first as a whole before letting the media to enter it.
2. Maintaining Suitable Environment for Microbial Growth:
There are various factors which influence the growth of the microorganisms. This includes the composition of the growth medium, concentration of salts, pH, temperature, level of aeration, etc. Most of the bacteria grow at neutral pH, whereas yeast and fungi prefer acidic pH.
3. Aeration and Mixing:
Mixing of the broth is essential for the uniform distribution of the nutrients in the culture medium. Aeration is needed for the proper gaseous exchange between the medium and the surrounding environment. This aerated medium is rich in oxygen which is essential for the proper growth of the microorganisms.
Aeration and mixing is achieved by shaking the medium on a shaker in the case of small scale cultures whereas in big bioreactors it is achieved by stirring the medium with the help of a mechanical stirrer with baffles attached to it.