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The basic processes for rDNA fermentation and purification normally include the following steps:
1. Inoculum Preparation: The aim is to develop for the production stage fermentation a pure inoculum in sufficient volume and in the fast-growing (logarithmic) phases so that a high population density is obtained. This is accomplished through a seed fermentation train.
2. The Medium: This is designed to provide the microorganism with all the nutrients it requires. Provision is usually made to add nutrients during fermentation.
3. Oxygen Supply: An adequate supply of oxygen is required. As oxygen is only slightly soluble in water, a number of methods are used to make oxygen more readily available to the microorganisms in the broth, including sparging, mechanical agitators, and dispersion baffles in the fermentor tank.
4. Temperature Control: Heat is generated both by the metabolism of nutrients and by the power dissipated in stirring and has to be removed by controlled cooling. Tank jackets or internal coils are used to control temperature.
5. Antifoam Agents: Microbiological systems that are vigorously stirred and aerated usually produce foam. Excessive foam cannot be tolerated and so provisions have to be made for adding antifoam agents.
6. Harvesting: This is the removal of the cells from the broth. This can be accomplished by cross-flow filtration or centrifugation.
7. Cell Lysis: With E. coli fermentations, the product protein is contained within the cell in the form of an inclusion body. High-pressure homogenizers are often used to chop up the E. coli bacteria into fine fragments, liberating the inclusion bodies for further processing.
8. Purification: This is the separation of the desired product from the other constituents in the harvested broth. Various processes including refolding, ultrafiltration/diafiltration, centrifugation, and chromatographic columns are employed to purify the product.
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